Vol. 5 núm. 2 - Sociedad Española de Microbiología

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Vol. 5 núm. 2 - Sociedad Española de Microbiología
Volumen 5. N.° 2
Septiembre 1989
ISSN 02 13-4101
PUBLICACIÓN DE LA SOCIEDAD
ESPAÑOLA DE MICROBIOLOGÍA
Microbiología
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Las gráficas crecimiento/tiempo permiten la comparacic
entre distintos medios y su óptima elección
Hace algunos años COULTER introdujo el EPICS-C, el citónnetro
de flujo más pequeño y fácil de utilizar. Después de 7 años de
experiencia y más de 5 0 0 instalaciones en todo el mundo,
COULTER se supera introduciendo el EPICS-CS: er«cell sorter»
más rápido, sencillo, compacto y económico del mercado, que
une a las características del EPICS-C una mayor capacidad de
memoria y análisis, impresora gráfica de alta resolución y gran
sencillez de manejo.
Si todavía piensa que la Citometría de Flujo es una técnica cara para su laboratorío,
CONSÚLTENOS.
.(JuLl tK
Si Ud. cree que la automatización
disminuye su propio control...
...Sistema Pasco para ID/CIVII de
DIFCO
desarrollado por y para microbiólogos.
que automatiza sos propias decisiones
U d . m i s i n o : establece las decisiones sobre el punto final de las diferentes
reacciones.
INOCULADOR
U d . m i s m o : controla la información sobre la susceptibilidad, con una completa flexibilidad y fácil interpretación.
VERSATILIDAD
desechable de 104 pocilios. Sin
ajuste de turbidez del inoculo.
Proceso de datos diseñado por y para microbiólogos
de gran versatilidad. Ordenador NCR de gran capacidad para almacenamiento de datos.
PAWELES
Panel de 104 pocilios. Análisis de 33 agentes antimicrobianos. No requiere rehidratación.
DATOS
Dosificación recomendada en base
a niveles alcanzables en suero
o tejido blando.
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45l
280 mg. por vía
intravenosa (IV) o
intramuscular (IM),
Cada 8 horas
Dosis de
4 mg. por kg
^^^—^
FRANCISCO SORIA MELGUIZO, S, A.
Caramuel. 38 - Tfno. 464 94 50 - 464 36 00 - Telex 43766 FSOR E - ?801 1 MADRID
MICROBIOLOGÍA SEM
Publicación de la Sociedad Española de Microbiología
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Manuel Benjamín Manzanal, Departamento
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Antonio Ventosa, Departamento de Microbiología, Facultad de Farmacia, Universidad de
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Seeberg, E., Nissez-Meyer, J. and Strike, P. (1976). den V gene of bacteriophage T4 determines
a DNA glycosilate specific for pyrimidine dimers in DNA. J. Viriol. 35, 790-797.
Tomasz, A. (1984). Building and breaking in the cell wall of bacteria - The role for autolysins.
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CONTENTS
Page
Transcription in vaccinia virus. Kent, R
Yeast species involved in spontaneous fermentation of Ribeiro wine. Cansado, J., Longo, E., Agrelo, D.
and Villa, T. (*)
Ultrastructure of the intracellular development of the bacteriophage 0C31. Rodriguez, A., Hardisson,
C and Suárez, /. E. (*)
Prevalence of Salmonella serotypes isolated in Spain from human and non human sources (1983-1987).
Echeita, M. A (*) and Usera, M. A
Selected characteristics of several strains of Lactobacillus plantarum. Mayo, B., Hardisson, C. and Braña,A.E(*)
The effect of Trichoderma viride C-1 UV mutagenization on cellulases activity. Witkowska, D., Bien, M.
and Sobiezczanski, J. f*j
Saprophytic fungi on hair and feathers from apparently healthy animals. Abarca, M." L. (*), Bragulat,
Mf R., Bruguera, Mf T and Cabanes, E J.
A rational proposal for plasmid nomenclature. Campos, R (*) and Martin-Luengo, E
69
79
89
95
105
113
121
127
(*) Corresponding author.
INDICE
Página
Transcripción en virus vaccinia. Kent, R
Levaduras asociadas a procesos de fermentación espontánea en vinos de Ribeiro. Análisis del homo/heterotalismo y sistema killer de las cepas de S. cerevisiae. Cansado, J., Longo, E., Agrelo, D. y
Villa, TC^)
Ultraestructura del desarrollo intracelular del bacteriófago 0C31. Rodríguez, A., Hardisson, C y Suárez,J.E.(*)
Prevalencia de los serotipos Salmonella de origen humano y no humano aislados en España (1983-1987).
Echeita, M. A (*) y Usera, M. A
Características relevantes de varias cepas de Lactobacillus plantarum. Mayo, B., Hardisson, C y Braña,A.E(*)
Efecto de la mutagenesis con UV sobre las actividades celulásicas de Trichoderma viride C-1. Witkowska, D., Bien, M. y Sobiezczanski, J. (*j
Hongos saprofitos aislados a partir de pelo y plumas de animales aparentemente normales. Abarca,
M." L. (*), Bragulat, Mf R., Bruguera, Mf T. y Cabanes, E J.
Una propuesta racional para la nomenclatura de plásmidos. Campos, P. (*) y Martín-Luengo, E
(*) A quien debe dirigirse la correspondencia.
69
79
89
95
105
113
121
127
M I C R O B I O L O G Í A SEM
5 (1989), 69-77
MINIREVIEW
Transcription in vaccinia virus
Richard Kent
Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road,
Cambridge CB13QP U.K.
(Received June 6,1989)
Summary
Transcription of poxvirus genes is temporally regulated and takes place in the cytoplasm of the
infected cell. Virus-encoded enzymes produce capped, methylated and polyadenylated mRNA. The
virus transcriptional apparatus recognises promoter sequences within the virus genome which bear
little resemblance to either eukaryotic or prokaryotic promoter regions. mRNA's produced late in
infection possess 5'poly (A) sequences which are not encoded in the genome. The process of transcription in poxviruses is reviewed using vaccinia virus as the prototype.
Key words: Poxvirus, vaccinia, transcription.
Resumen
La transcripción de los genes de poxvirus está regulada a lo largo del ciclo de multiplicación y
tiene lugar en el citoplasma de la célula infectada. Las enzimas codificadas por el virus producen
m-RNA poHadenilados, metilados y con «cap». El aparato transcripcional del virus reconoce las secuencias promotoras contenidas en el genoma vírico que muestran poca semejanza con las regiones
promotoras eucarióticas o procarióticas. Los m-RNAs producidos en la fase tardía de infección
contienen secuencias 5' poli (A) que no están codificadas en el genoma. El proceso de transcripción
en poxvirus se estudia utilizando como prototipo el virus vaccinia.
Introduction
Vaccinia is the prototypical and most studied member of the orthopoxvirus genus (reviewed in
45). Other members of this genus include buffalopox, camelpox, cowpox, ectromelia and variola.
All of these viruses are closely related, both genetically (37) and antigenically (23). The latter relationship has allowed both cowpox and vaccinia to be used for successful prophylactic immunisation
against variola (smallpox) infection (reviewed in 25). Although the origin of vaccinia virus is unknown, it is clear from genetic analysis that it is unlikely to have arisen from simple mutation of, or
recombination between, either variola or cowpox viruses (37).
Vaccinia has an ovoid virion of over 200 nanometers in length and a complex morphology. The
majority of virions remain intracellular and are surrounded by a single lipoprotein bilayer. Extracellular virions, which are probably of greater importance in the in vivo spread of infection (51), have
an additional outer lipoprotein envelope which is acquired from the Golgi membranes of the host-
70
TRANSCRIPTION IN VACCINIA VIRUS
cell (30). Within the inner envelope of the virion is a biconcave core which contains virus DNA and
many enzymes (reviewed in 45). Among these core-associated enzymes are a DNA-dependent
RNA polymerase (1) and enzymes capable of capping, methylating and polyadenylating mRNA
(41, 46). Within the concavities of each vaccinia virion core lie two structures of unknown function
called lateral bodies.
The vaccinia genome is a linear double-stranded DNA molecule of over 180 kilobase pairs
whose two strands are covalently linked by single-stranded hairpin loop structures (2). The genome
possesses an inverted terminal repetition of about 10 kilobase pairs within which are short, direct
tandem repeats (76). The central portion of the orthopoxvirus genome is well conserved between
different members of the genus; variation occurs primarily at the ends of the genome (37).
Replication of the virus genome occurs in the cytoplasm of the host-cell and is believed to involve a self-priming strand displacement mechanism by a virus-encoded DNA polymerase (22) following the introduction of nicks close to the termini of the genome.
Knowledge of the vaccinia genome and its structure has been obtained from analysis of restriction endonuclease fragments of virus DNA (20, 37), transcriptional analysis (e. g. 18) and more recently from nucleotide sequence analysis of large segments of the genome (e. g. 49, 52, 56, 57, 68,
72). These studies have revealed that vaccinia mRNA's are coUinear with their genes which are closely packed, sometimes overlapping with each other. Transcription occurs in each direction of both
strands of the genomic DNA.
Poxvirus gene expression is temporally regulated. Immediately upon infection transcription
early genes are expressed. After the onset of DNA replication transcription of most early genes is
suppressed and expression of the late genes begins. The control of expression of these two major
classes of genes will be discussed in some detail (see below). Recently a novel class of vaccinia gene
has been described (69) which is expressed after DNA replication has begun but before the expression of the majority of the late genes. At present little more is known about these intermediate genes: they will not be discussed further in this review.
Interest in vaccinia virus and the regulation of poxvirus gene expression has been stimulated
by the recent development of recombinant vaccinia viruses which have been used to express a variety of foreign genes in eukaryotic cells (38). Such viruses can infect a wide range of mammalian
cell types and foreign DNA sequences of up to 25 kilobase pairs in length can be stably integrated
into non-essential regions of the vaccinia genome, such as the thymidine kinase gene, without loss
of virus infectivity (62). Recombinant vaccinia viruses have a number of possible applications, they
can be considered as candidate live vaccines with potential veterinary and medical applications
(e. g. 7, 47, 63) and as a means of investigating the immune response (e. g. 34) or protein function
(e. g. 24). However, the expression of foreign genes in cells infected with recombinant vaccinia viruses is dependent upon the presence of poxvirus transcriptional control sequences upstream of
the heterologous open reading frames (39).
Methods of investigating transcription
At the same time as heightening interest in poxvirus transcription the development of vaccinia
as an expression vector has also provided a powerful technique for its study. For example, putative
promoter elements can be isolated, manipulated and reintroduced into the vaccinia genome upstream of a gene with an easily assayable product. The effect of any particular mutation on the activity of that promoter region can be assessed by assaying the level of the reporter gene product in
cells infected with the recombinant virus. Vaccinia virus promoters which drive the expression of
non-essential genes can be manipulated and reintroduced into their normal position within the genome (e. g. 19). However a similar technique can be employed for any promoter sequence. The
R. KENT
71
promoter sequence is maintained at its original position while a copy, or mutated derivative, is introduced into a non-essential region of the genome upstream of a suitable reporter gene. This method has been used to identify important regulatory sequences in both early (e. g. 74) and late (e. g.
4) vaccinia promoters. The amount and kinetics of transcription from unmutated translocated promoter sequences are similar to those observed from the same sequences at their authentic genomic
position.
A more rapid technique for studying late promoter activity without the need to construct recombinant viruses is the vaccinia virus-dependent transient expression system (15). Plasmids containing a vaccinia late promoter element are transfected into vaccinia-infected cells. The transfected
promoter element is recognised by the vaccinia transcriptional apparatus in the infected cell and the
reporter gene is expressed. The level of expression of the reporter gene provides an estimate of promoter strength. The expression of the reporter gene is dependent upon the presence of an upstream
vaccinia late promoter; neither vaccinia early promoters nor eukaryotic promoters are effective.
The inactivity of vaccinia early promoters in this system is presumed to be due to the close association of the early transcriptional apparatus with the virion cores during the early stages of infection.
The relative activities of late promoters in this system are consistent with those observed in cells infected with the corresponding recombinant viruses (73).
Extracts of vaccinia virus particles (28, 54) or vaccinia-infected cells (26, 53) will transcribe
DNA from early vaccinia virus promoters. Such systems have provided a means of identifying some
of the factors involved in transcription (13, 60) including a transcription factor which interacts with
vaccinia early promoter regions in electrophoretic mobility shift assays and DNase I footprint
analysis (78). An extract of vaccinia-infected cells has also been shown to be capable of transcribing
DNA from late vaccinia promoters (58, 77).
Transcription
The majority of DNA-containing viruses replicate within the nucleus of the infected cells.
Transcription of virus genes is frequently performed by host-cell RNA polymerase II and initiates
downstream of regions within the virus genome which show sequence homology to cellular RNA
polymerase II promoters (e. g. 3, 16, 71). Vaccinia virus, however, rephcates exclusively within the
cytoplasm of infected cells and encodes many of the polysephides required for transcription of its
genes (1,32, 64).
The vaccinia DNA-dependent RNA polymerase shares a number of features with eukaryotic
class II RNA polymerases. Both are multipartite enzymes of about 500 kiloDaltons with two large
subunits of over 100 kiloDaltons as well as a number of smaller components. The eukaryotic enzyme has at least eight of these smaller polypeptides which range in size from 40 to 10 kiloDaltons.
Vaccinia RNA polymerase has at least seven subunits of less than 40 kiloDaltons which, hke the
two larger polypeptides, are encoded within the central conserved region of the vaccinia genome
(32). The nucleotide sequence of the gene which encodes the 147 kiloDaltons component of the
vaccinia polymerase has been determined and reveals regions of extensive honiology with the largest subunits of eukaryotic RNA polymerases II and III, and slightly less homology with the prokaryotic RNA polymerase (11). Like its eukaryotic counterpart, the purified vaccinia enzyme is
unable to transcribe double-stranded DNA in the absence of protein extracts of virions or virus-infected cells (26,28, 53, 54).
Vaccinia transcripts have 5' cap structures, 7methyl-G-p-p-p-(A/G)-p-RNA, similar to those of
normal eukaryotic mRNA's. Nascent transcripts are processed by an enzyme complex of over 100
kiloDaltons. The enzyme consists of at least two subunits, the largest of which has been shown to be
virus encoded (43). The enzyme has a series of activities; RNA triphosphatase, guanylyltransferase
72
TRANSCRIPTION IN VACCINIA VIRUS
and guanine-7-methyltransferase (41, 70) and may also perform a function in the termination of
early vaccinia transcription (see below).
Despite its cytoplasmic replication, vaccinia is dependent on the host-cell nucleus for productive infection; maturation of vaccinia virus is blocked in enucleated cells (31). This dependence may
result from the need of vaccinia to selectively recruit components of the nucleus for successful virus
maturation (reviewed in 48). One of these components appears to be the largest subunit of RNA
polymerase II. This nuclear polypeptide is translocated to the cytoplasm of cells infected with rabbit
poxvirus and copurifies with rabbit poxvirus RNA polymerase isolated from virions (44).
Further evidence for the involvement of cellular RNA polymerase II in the replication comes
from studies involving an inhibitor of the enzyme, a-amanitin (to which the virus RNA polymerase
is relatively resistant); the usual inhibition of poxvirus replication by a-amanitin can be by-passed
in cells which are resistant to the effects of the drug (61). The inhibition of poxvirus replication by
a-amanitin can also be by-passed by mutation of the virus. The region of the genome which encodes the gene(s) responsible for this resistance has recently been sequenced (65). However, the mechanism through which virus a-amanitin resistance is acquired, and exact nature of the role of the
host-cell nucleus in vaccinia replication, have yet to be determined.
Transcription of early genes
Following virus adsorption and penetration the virus cores are uncoated and released into the
cytoplasm of the host-cell. Transcription of about 100 genes, distributed throughout the virus genome, begins immediately. These genes, transcribed prior to DNA replication, are designated «early».
The cores contain all the enzymes necessary to generate capped (41), methylated (70) and polyadenylated (46) transcripts of the early class of genes. Consequently, transcription of early genes is
not affected by inhibitors of either protein or DNA synthesis. Virus cores are capable of transcribing these genes in cell-free systems (17). Many of the enzymes involved in this transcription, including components of the virus RNA polymerase and the mRNA capping enzyme, are virus encoded
(32, 43). As might be expected for a virus which replicates within the cytoplasm, there is no evidence for splicing of vaccinia virus early transcripts and the 5' terminus of the mRNA is believed to correspond to the transcriptional start site.
Transcription of each early gene initiates at a single discrete site both ¿n vivo (36) and in cellfree systems (53). Eukaryotic RNA polymerase II promoters have conserved sequence motifs upstream of the transcriptional start sites. These are the TATA or Goldberg-Hogness box and the
CAAT sequence which normally occur about 30 and 70 nucleotides (respectively) upstream of the
transcriptional start site. Despite the similarities between the vaccinia RNA polymerase and the eukaryotic class II polymerases, examination of the nucleotide sequences upstream of virus early
transcriptional start sites reveals no homology to known eukaryotic (or prokaryotic) promoter elements. This is consistent with the observation that cell-free extracts of uninfected cells, unlike infected-cell extracts, are incapable of transcribing vaccinia DNA (53).
Like prokaryotic and eukaryotic RNA polymerase Il-mediated transcription, expression of
vaccinia early genes is controlled by sequences upstream of the mRNA start sites. Temporally controlled expression of foreign genes in recombinant vaccinia viruses can be obtained when the foreign open reading frame is placed just downstream of the transcriptional start site of a vaccinia
virus early gene (e. g. 19). Deletion mutagenesis of such early promoter regions has demonstrated
that the sequences which control transcription can be contained within a short region, less than 40
base pairs, upstream of the RNA start site (10,14,19, 74). Vaccinia virus genes are closely packed
within the genome, therefore, in some cases, the early promoter of one gene lies within of open reading frame of another.
R. KENT
73
TABLE 1
MINIMUM SEQUENCE REQUIREMENTS FOR CORRECT TEMPORAL REGULATION OF
TRANSCRIPTION FROM VACCINIA PROMOTERS
The 5'flankingsequences from several vaccinia genes have been investigated using deletion mutagenesis. The
sequences shown have been shown to correctly regulate expression of foreign genes in recombinant viruses.
The major RNA start sites are marked by asterisks (*) and the initiation codons for late genes are underlined
(ATG).
a) EARLY PROMOTER SEQUENCES
Gene
Reference
TK
CGAATAAAGTGAACAATAATTAATTCTTTATTGTC
74
7.5kDa
CGTAAAAGTAGAAAATATATTCTAATTTATTGCA
14
b) LATE PROMOTER SEQUENCES
Gene
Reference
llkDA
GTTTTTTTCTATGCTATAAATG
28KDa
CACAAAAAAAACTTCTCTAAATG
5
75
Vaccinia early promoters contain conserved sequence motifs:
TAAA-N0.4.TAAAAA-N0.2-TAA-A/T-A/T
which are centred approximately 20 nucleotides upstream of the transcriptional start sites (40, 66).
These motifs have been demonstrated to interact with at least one early transcription factor to
allow initiation of transcription by the virus RNA polymerase. This early transcription factor is probably a heterodimer of 82 and 77 kiloDalton subunits and has been demonstrated to have a DNAdependent ATPase activity (12, 13). Mutations which decrease the ability of an early vaccinia
promoter to bind this factor decrease the transcriptional activity of that promoter region (78).
Early transcription terminates at discrete sites about 50 base pairs downstream of a signal sequence TTTTTNT, or, as it occurs within the nascent transcript, UUUUUNU (55). This termination is dependent upon another transcription factor associated with the RNA polymerase and
which copurifies with, and may be identical to, the vaccinia mRNA capping enzyme (60).
Folyadenylation of vaccinia mRNA's, unlike that of cellular transcripts, does not appear to require any specific signal sequences as prematurely terminated mRNA's possess poly (A) tails (27).
Between two and five hours after infection with vaccinia virus, replication of the virus genome
begins. At the same time there is a profound change in the pattern of transcription and gene expression. Most of the early virus proteins are not produced after DNA replication although in vitro
translation of RNA from infected cells suggests that significant amounts of functional early mRNAs
are still present at this time (67). Prior to DNA replication about half the vaccinia genome, corresponding to the early genes, is transcribed. After DNA synthesis the whole of the genome is transcribed (8). A new class of genes is expressed from these transcripts; these genes are designated «late
genes». The factors controlling this switch in gene expression are largely unknown although it can
be prevented by inhibitors of DNA synthesis.
IVanscription of late genes
Nuclease protection experiments which map the 5'termini of late vaccinia mRNA's suggest
that transcription of each late gene initiates at a discrete site which is usually just upstream of the
74
TRANSCRIPTION IN VACCINIA VIRUS
start of the open reading frame (e. g. 35, 56, 57). Examination of the sequences which surround these late transcriptional start sites reveals no homology to previously observed transcriptional control
signals, not even those of the vaccinia virus early promoters.
Late vaccinia gene expression is regulated by short sequences preceding and including the
transcriptional start site. High level expression of heterologous open reading frames in vaccinia-infected cells can be directed from short sequences which normally precede late open reading frames
(4, 5,14,29,42,75). The length of these late promoter sequences can be less than 20 base pairs.
Few areas of sequence homology between different late promoter elements have been reported. The most obvious conserved sequence is the motif TAAAT which occurs at the transcriptional
start site of a number of late vaccinia genes. Frequently this motif overlaps with the translational
start site, resulting in the motif TAAATG, and has been proposed to play a crucial role in the initiation of late transcription (56). Support for this proposal has come from the results of mutagenesis
experiments involving the deletion of, or multiple base pair substitutions within, the area of this
motif. Both of these procedures abolish the promoter activity of the sequence which precedes the
11 kiloDalton polypeptide open reading frame (5, 29). In addition, ¿n vitro transcription from this
promoter in extracts of vaccinia-infected cells is abolished by conversion of the motif into the sequence TGAAT. The introduction of a similar mutation into the TAAAT motif of another late promoter, from a 28 kiloDalton polypeptide gene, has been reported to abolish its ¿n vivo activity (77).
Those vaccinia virus early genes which continue to be expressed at late tinies possess upstream
sequences which are characteristic of both early and late promoter elements. Prior to DNA replication, transcription occurs from the early promoter region; after DNA replication, transcription initiates from within the late promoter element (14).
One peculiar characteristic of late poxvirus transcripts, whether generated in vivo or in vitro, is
the presence of 5' poly (A) sequences for up to 35 nucleotides upstream of the start of the mRNA
as defined by SI or mung bean nuclease protection experiments (6, 21, 50, 58, 59, 77). The 5' termini of the poly (A) heads generated in vivo are capped and methylated (59).
The mechanism by which these poly (A) heads are generated is unclear. In vitro experiments (58) are consistent with a model in which transcription initiates at the second residue of
the TAAAT motif but in which the transcriptional complex repeatedly slips in a 3'-5' direction before progressing from the position of the motif. A similar mechanism has been proposed to account for 5'poly (A) sequences generated on in vitro transcripts from bacteriophage T4 late
promoters (33). However, it is not yet possible to exclude the possibility that transcription is initiated from a poly (A) primer. The biological functions of the poly (A) heads are unknown.
There has been one report (6) which suggests that the 5' poly (A) sequences of some late transcripts may be preceded by longer sequences, perhaps several thousand nucleotides long, which are
derived from transcription of separate regions of the vaccinia genome. However, this observation
has not been confirmed by other workers and may have been an artefact resulting from infidelity of
the reverse transcriptase during cloning procedures.
Another unusual feature of late poxvirus transcription is the lack of discrete termination.
Transcripts arising from a late promoter may continue for several kilobases beyond the end of the
first open reading frame (18). Early transcription termination signals are not recognised late in infection. This phenomenon accounts for the presence of large amounts of double-stranded RNA
present in cells late in infection wich vaccinia viruses (9).
Lack of precise termination of transcription does not appear to be universal for late poxvirus
genes. Patel & Pickup (50) report that transcripts of the 160 kiloDalton polypeptide gene of cowpox
virus are a defined length. Since the gene is not close to either of the termini of the genome this finding suggests that the normal 3'terminal heterogeneity of late messages does not arise from a failure of the RNA polymerase to recognise termination signals, but rather to an absence of such signals
at the 3'terminus of most late genes.
R. KENT
75
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18. Cooper, J. A., Wittek, R. and Moss, B. (1981). Extension of transcriptional and translational map of the left end of the
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20. DeFilippes, F. M. (1982). Restriction enzyme mapping of vaccinia vius DNA. J. Virol. 43,136-149.
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23. Espósito, J. J., Obijeski, J. F. and Nakano, J. H. (1977). Serological relatedness of monkeypox, variola and vaccinia
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24. Falkner, F G., Fuerst, T. R. and Moss, B. (1988). Use of vaccinia virus vectors to study the synthesis, intracellular location
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25. Fenner, F (1985). Poxviruses. In: B. N. Fields, R. J. Chanock and B. Roizman (eds.). Virology Raven Press (New York).
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virus-infected HeLa cells. J. Virol. 53,822-826.
27. Gershowitz, A. and Moss, B. (1979). Abortive transcription products of vaccinia virus are guanylylated, methylated and
polyadenylated. J. Virol. 31,849-853.
28. Golini, F and Kates, J. R. (1984). Transcriptional and translational analysis of a strongly expressed early region of the
vaccinia virus genome. J. Virol. 49,459-470.
29. Hangii, M., Bannwarth, W and Stunnenberg, H. G. (1986). Conserved TAAAT motif in vaccinia virus late promoters:
overlapping TATA box site and site of transcriptional initiation. The EMBO J. 5,1071-1076.
76
TRANSCRIPTION IN VACCINIA VIRUS
30. Hiller, G. and Weber, K. (1985). Golgi derived membranes that contain acylated viral polypeptide are used for vaccinia
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32. Jones, E. V, Puckett, C. and Moss, B. (1987). DNA-dependent RNA polymerase subunits encoded within the vaccinia virus genome. J. Virol. 61,1765-1771.
33. Kassavetis, G. A., Zentner, P. G. and Geiduschek, E. (1986). Transcription at bacteriophage T4 variant late promoters.
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14256-14265.
34. King, A. M. Q., Stott, E. J., Langer, S. J., Young, K. K.-Y, Ball, L. A. and Wertz, G. W. (1987). Recombinant vaccinia viruses carrying the N gene of respiratory syncytial virus: studies on gene expression in cell culture and immune response
in mice. J. Virol. 61,2885-2890.
35. Lee-Chen, G. J. and Niles, E. G. (1988). Map positions of the 5' ends of eight mRNA's synthesised from late genes in the
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36. Lee-Chen, G. J., Bourgeois, N., Davidson, K., Condit, R. C, and Niles, E. G. (1988). Structure of the transcription initiation and termination sequences of the seven early genes in the vaccinia virus Hind III D fragment. Virology 163,64-79.
37. Mackett, M. and Archard, A. C (1979). Conservation and variation in orthopoxvirus genome structure. J. Gen. Virol. 45,
683-701.
38. Mackett, M. and Smith (1986). Vaccinia virus expression vectors. J. Gen. Virol. 67,2067-2082.
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39. Mackett, M., Smith, G. L. and Moss, B. (1984). General method for production and selection of vaccinia virus recombinants expressing foreign genes. J. Virol. 49,857-864.
40. Mars, M. and Beaud, G. (1987). Characterisation of vaccinia virus early promoters and evaluation of their informational
content. J. Mol. Biol. 198,619-631.
41. Martin, S. A., Paoletti, E. and Moss, B. (1975). Purification of mRNA guanylyltransferase and mRNA (guanine-7-)
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42. Miner, J. N., Weinrich, S. L. and Hruby, D. E. (1988). Molecular dissection of cisacting regulatory elements from 5'-proximal regions of a vaccinia late gene cluster. J. Virol. 62,297-304.
43. Morgan, J. R., Lawrence, K. C. and Roberts, B. E. (1984). Identification of the DNA sequences encoding the large subunit of the mRNA capping enzyme of vaccinia virus. J. Virol. 52,206-214.
44. Morrison, D. K. and Moyer, R. W. (1986). Detection of a subunit of cellular pol 11 within highly purified preparations of
RNA polymerase isolated from rabbit poxvirus virions. Cell 44,587-596.
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46. Moss, B., Rosenblum, E. N. and Gershowitz, A. (1975). Characterization of a polyriboadenylate polymerase from vaccinia virions, J. Biol. Chem. 250,4722-4729.
47. Moss, B., Smith, G. L., Gerin, J. L. and Purcell, R. H. (1984). Live recombinant vaccinia virus protects chimpanzees
against hepatitis B. Nature 311,67-69.
48. Moyer, R. W. (1987). The role of the host cell nucleus in vaccinia virus morphogenesis. Virus Res. 8,173-191.
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of the 16 kb vaccinia virus Hind III D fragment. Virology 153,96-112.
50. Patel, D. D. and Pickup, D. J. (1987). Messenger RNAs of a strongly expressed late gene of cowpox virus contain 5'-terminal poly(A) sequences. The EMBO J. 6,3787-3794.
51. Payne, L. G. (1980). Significance of extracellular enveloped virus in the in vitro and in vivo dissemination of vaccinia. J.
Gen. Virol. 50,89-100.
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late genes in a conserved segment of the vaccinia virus genome. Nucl. Acids. Res. 13,985-998.
53. Puckett, C. and Moss, B. (1983). Selective transcription of vaccinia virus genes in template dependent soluble extracts of
infected cells. Cell 35,441-448.
54. Rohrmann, G. and Moss, B. (1985). Transcription of vaccinia virus early genes by a template-dependent soluble extract
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55. Rohrmann, G., Yuen, L. and Moss, B. (1986). Transcription of vaccinia virus early genes by enzymes isolated from vaccinia virions terminates downstream of a regulatory sequence. Cell 46,1029-1035.
56. Rosel, J. L. Earl, P. L., Weir, J. P. and Moss, B. (1986). Conserved TAAATG sequence at the transcriptional and translational start sites of vaccinia virus late genes deduced by structural and functional analysis of the Hind III H genome fragment. J. Virol. 60,436-449.
57. Schmitt, J. R and Stunnenberg, H. G. (1988). Sequence and transcriptional analysis of the vaccinia virus Hind III I fragment. J. Virol. 62,1889-1897.
R. KENT
77
58. Schwer, B. and Stunnenberg, H. G. (1988). Vaccinia virus late transcripts generated in vitro have a poly(A) head. The
EMBO J. 7,1183-1190.
59. Schwer, B., Visca, R., Vos, J. C. and Stunnenberg, H. G. (1987). Discontinuous transcription or RNA processing of vaccinia vkus late messengers results in a 5'-poly(A) leader. Cell 50,163-169.
60. Shuman, S., Broyles, S. S. and Moss, B. (1987). Purification and characterisation of a transcription termination factor
from vaccinia virions. J. Biol. Chem. 262,12372-12380.
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62. Smith, G. L. and Moss, B. (1983). Infectious poxvirus vectors have the capacity for at least 25,000 base pairs of foreign
DNA. Gene 25,21-28.
63. Smith, G. L., Murphy, B. R. and Moss, B. (1983). Construction and characterisation of an infectious vaccinia virus recombinant that expresses the influenza haemagglutinin gene and induces resistance to influenza virus infection in hamsters.
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65. Tamin, A., Villareal, E. C , Weinrich, S. L. and Hruby, D. E. (1988). Nucleotide sequence and molecular genetic analysis
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68. Venkatesan, S., Gershowitz, A. and Moss, B. (1982). Complete nucleotide sequence of two adjacent early vaccinia virus
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M I C R O B I O L O G Í A SEM
5 (1989), 79-88
Levaduras asociadas a procesos de fermentación espontánea
en vinos de Ribeiro. Análisis del homo/heterotalismo y sistema
killer de las cepas de S. cerevisiae
José Cansado, Elisa Longo, Dolores Agrelo y Tomás G. Villa *
Departamento de Microbiología. Facultad de Farmacia. Campus Universitario. Universidad de Santiago de Compostela. Spain.
(Recibido febrero 15/abril 12,1989
Summary
A taxonomic analysis has been carried out concerning the different yeast species involved in
spontaneous fermentations of Ribeiro wine. The results revealed the presence of 15 species belonging to 7 genera: Saccharomyces, Torulaspora, Dekkera, Saccharomycodes, Debaryomyces, Kloeckera and Candida. Within the genus Candida a high number of species was detected. The 28 isolated strains of S. cerevisiae showed with some exceptions, a remarkable sporulation ability and viability of the meiotic products. All the strains were homothaUic. Among all S. cerevisiae strains, 6 exhibited killer K2 activity with higher isolation percentages in intermediate fermentations than in late
fermentations.
Key words: Wine, yeasts, sporulation, killer activity.
Resumen
Se ha llevado a cabo un estudio taxonómico de las poblaciones de levaduras implicadas en fermentaciones espontáneas de vinos de Ribeiro. Los resultados indicaron la presencia de 15 especies
englobadas en siete géneros: Saccharomyces, Torulaspora, Dekkera, Saccharomycodes, Debaryomyces, Kloeckera y Candida. Dentro del género Candida se encontró un elevado número de especies.
Las 28 cepas de S. cerevisiae aisladas mostraron, salvo excepciones, una notable capacidad de esporulación y viabilidad de los productos meióticos. En todos los casos se trató de cepas homotálicas. Se detectaron seis cepas de S. cerevisiae con actividad killer K2 con porcentajes de aislamiento
más elevados en fases intermedias que en fases finales de fermentación.
Introducción
La identificación y clasificación de las especies de levaduras que intervienen en la fermentación de un mosto resulta de gran utilidad en la industria vitivinícola, ya que permite conocer las posibilidades enológicas reales de un vino en función de la microflora propia de dicho mosto. El estudio de la dinámica poblacional de levaduras y su distribución durante las sucesivas etapas del proceso fermentativo puede, a su vez, contribuir a resolver los problemas que a menudo se plantea la
industria de vinificación (17).
(*) A quien debe dirigirse la correspondencia.
80
LEVADURAS ASOCIADAS A PROCESOS DE FERMENTACIÓN...
En general puede afirmarse que la flora blastomicética implicada en las fermentaciones espontáneas del mosto está representada, en las diferentes comarcas vitivinícolas, por unas 100 especies
de levaduras. Su distribución en la viña y en la bodega, así como su evolución en las fermentaciones, responde a un esquema común (16).
En Galicia existen aportaciones descriptivo-taxonómicas importantes en el campo de las levaduras con interés enológico y que cubren diferentes comarcas vitivinícolas [Longo, E., Agrelo, D. y
Villa, T. G. (1987). XI Congr. Nac. Microbiol, pp. 866, 868; 9,15].
En levaduras industriales en general y en vínicas en particular la mayoría de las cepas de S. cerevisiae responsables del proceso fermentativo son homotálicas (21), siendo este carácter bien conocido desde el punto de vista genético (8). Su presencia complica el análisis y estudio de características susceptibles de manipulación genética.
Por otra parte, el fenotipo killer se encuentra ampliamente distribuido en levaduras, incluyendo las especies del género Saccharomyces. En S. cerevisiae se han descrito diversos tipos de carácter killer, destacando el Ki y el K2. El Kj fue el originariamente descrito (12) y se encuentra
principalmente en cepas de laboratorio, mientras que el K2 se describió en cepas vínicas (13). Las
cepas portadoras del fenotipo killer se caracterizan por secretar una toxina de naturaleza glicoproteica que mata a las cepas sensibles pero no a sí mismas y está asociado a la presencia en el citoplasma de ds-RNA (4).
En el presente trabajo se describe el aislamiento e identificación de levaduras vínicas asociadas
a fermentaciones espontáneas de vinos de Ribeiro, así como la sucesión de poblaciones que se establece durante las diferentes fases del proceso de fermentación. Además se tuvo también en cuenta
el carácter killer, homo/heterotalismo, porcentaje de ascosporulación y viabilidad de los productos
meióticos, en base a la realización de una caracterización de las cepas de S. cerevisiae implicadas en
las fermentaciones que permitiese la selección de aquellas con mayor potencialidad de manipulación genética.
Materiales y métodos
Método de maestreo
El muestreo se llevó a cabo en dos bodegas representativas de la comarca objeto de estudio en
cuanto a las variedades de uva empleadas y a la metodología de vinificación. Las cubas 1, 2 y 3 corresponden a vino blanco, mientras que la 4 es de vinificación en tinto. Los depósitos de fermentación fueron en todos los casos de cemento recubiertos con resina Epoxi, con volúmenes de 25.000 1
por depósito. La uva de vinificación fue Jerez en un 95 % en cuanto a los vinos blancos y 100 % de
Alicante en los tintos. El mosto entró en las cubas con una densidad media de 1.075 g/1 y pH 3,2; fue
sulfitado en las cubas 1 y 2 con 80 mg/1 de metabisulfito potásico y con 200 mg/1 en las cubas 3 y 4,
siendo la temperatura media de las bodegas de 19° C.
Durante los sucesivos períodos de fermentación se recogieron asépticamente muestras de 100
mi a una altura media de la cuba de fermentación correspondiendo a mosto sulfitado, mosto en fermentación, mosto-vino y vino, transportándose rápidamente al laboratorio en recipientes refrigerados a una temperatura de 4° C.
Aislamiento de levaduras
Las muestras fueron diluidas o concentradas en función de la densidad celular. En el primer caso se efectuaron diluciones seriadas en agua destilada estéril (10"' a 10"^), mientras que en el según-
J. CANSADO, E. LONGO, D. AGRELO Y T. G. VILLA
81
do se procedió a la filtración a través de membranas de 0,22 jim que fueron incubadas sobre placas
Petri con medio YPD-agar (extracto de levadura, Difco, 1 %; peptona, Difco, 2 %; glucosa, Difco,
2 %; agar, Difco, 2 %) ajustado a pH de 5,7.
A partir de la dilución más conveniente en cada muestra, el aislamiento de levaduras se efectuó inoculando por extensión alícuotas de 0,1 mi sobre placas de Petri con medio YPD-agar. Después de 3 a 7 días de incubación a 25° C, la presencia de colonias de diferente morfología se hizo
evidente, por lo que se procedió a su aislamiento en placas con medio YM-agar (extracto de malta,
Difco, 0,3 %; extracto de levadura, 0,3 %; peptona, 0,5 %; glucosa, 1 %; agar, 2 %) ajustado a pH
5,7. Los cultivos puros así obtenidos se conservaron en tubo inclinado sobre medio de conservación
YPD a 4° C.
Análisis taxonómico
La identificación de las cepas aisladas se llevó a cabo según los criterios de Barnett y col. (3).
Morfología celular. Se realizó sembrando los cultivos en medio YM-agar y observando las características morfológicas en el microscopio de contraste de fases a los 3 ó 5 días de crecimiento a
25° C. Estas pruebas se completaron con la observación de la morfología de las colonias, así como
su textura y pigmentación.
Pruebas bioquímicas: Fermentación de glucosa; asimilación de nitrato y nitrito potásicos; asimilación de compuestos carbonados (pentosas: L-arabinosa, D-arabinosa, ribosa, xilosa; hexosas:
glucosa, galactosa, sorbosa; disacáridos: sacarosa, maltosa, celobiosa, trehalosa, lactosa, melibiosa;
trisacáridos: rafinosa, melezitosa: polisacáridos: almidón, inulina; alcoholes: glucitol, eritritol, manitol, mio-inositol; ácidos orgánicos: succínico, cítrico y láctico). Los diferentes medios de cultivo para
la asimilación de los compuestos carbonados contenían 0,67 % de YNB (Difco-yeast nitrogen base), agar al 2 % (Difco) y la fuente de carbono correspondiente (Merck) al 1 %. Para la asimilación
de nitrato y nitrito potásicos, el medio contenía 1,17 % YCB (Difco-yeast carbon base), agar al 2 %
(Difco) y 1 % de la fuente de nitrógeno (Merck).
Pruebas fisiológicas: Resistencia a la cicloheximida (Sigma: entre 0,01 y 0,1 %); resistencia a altas presiones osmóticas (YNB-glucosa al 50 y 60 %); resistencia a la temperatura (YNB-glucosa incubado a 37° C); reproducción asexual (simétrica o asimétrica); caracteres de reproducción sexual
(porcentaje de ascosporulación y número de esporas por asea. Las cepas se sembraron en agar-acetato (11) y agar-Gorodkova (10) a 30° C, observando en el microscopio de contraste de fases la presencia de aseas y ascosporas a los 3, 7,15 días y posteriormente cada 3 días hasta los 30); la formación de seudomicelio se observó sembrando las cepas en estría sobre portaobjetos con medio patata-glucosa-agar (10), colocados sobre un tubo en U dentro de una plaça de Petri con un papel de filtro impregnado con agua estéril.
Caracteres de crecimiento en mosto: Se realizaron en tubos Durham y con mosto natural estéril de Ribeiro. Los inóculos se adicionaron a una concentración final de 10^ cel/ml y se estudió la capacidad fermentativa, formación de velo, anillo y sedimento.
Las cepas se sembraron con un replicador automáico en grupos de 21. La lectura de los resultados se llevó a cabo después de 3, 7 y 15 días de incubación a 25° C, anotando la respuesta a cada ensayo en función de la ausencia o presencia de crecimiento, comparando éste con el obtenido en medio con 0,67 % YNB (Difco-yeast nitrogen base) y 2 % agar (Difco) utilizado como control (3).
Para confirmar la exactitud del método de identificación empleado y asegurar así la veracidad
de los resultados obtenidos se incluyeron cepas patrón de diferentes especies: Saccharomyces cerevisiae CECT 1170, Kluyveromyces marxianus CECI 1121, Candida utilis CECI 1061, Hansenula
anómala CECT 1107, Pichia carsoni CECT 1129, Kloeckera apiculata CECT 1120, y Saccharomycodes ludwigii CECT 1382.
82
LEVADURAS ASOCIADAS A PROCESOS DE FERMENTACIÓN...
Disección de aseas y determinación de homo/heterotalismo
Las cepas esporuladas de S. cerevisiae fueron tratadas con una solución de zimoliasa-5000 (Seikagaku Kogyo Co., Ltd. Japón) durante 1 ó 2 horas a 30° C y las ascosporas se aislaron por micromanipulación (7). En todos los casos se disecaron un mínimo de 6 aseas por cepa, determinando
posteriormente la viabilidad de los productos meióticos que a su vez se conjugaron con cepas a y a
heterotálicas de S. cerevisiae, observándose la formación de zigotos (20). Al mismo tiempo, en clones
derivados de ascosporas aisladas por micromanipulación se investigó su capacidad ascosporogénica.
Determinación del carácter killer
Las cepas a ensayar fueron sembradas sobre césped de la cepa sensible de S. cerevisiae 5 x 47
(a/a, hisl/+, trpl/+, ura3/+, K~) sembrada en medio YPD suplementado con azul de metileno (30
mg por cada 100 mi de medio) y tamponado a pH 4,5 con tampon citrato-fosfato 0,2M (12). Las cepas portadoras del fenotipo killer se manifestaron por la producción de un halo de inhibición en el
césped de la cepa sensible. Para determinar si el carácter killer observado en las cepas vínicas era
de tipo Ki o K2, se sembraron a su vez sobre céspedes de cepas de referencia: 1385 (a, lysl, ural,
K2^) y T158c (a his 4c-864, Kj"""^), teniendo en cuenta que las cepas portadoras del sistema killer Ki
inhiben el crecimiento de las K2 y viceversa (22).
Resultados
Muestreo
En total se tomaron 15 muestras correspondientes a mosto sulfitado, mosto en fermentación y
vino joven, de las cuales se aislaron 93 cultivos de levaduras, 82 en las cubas 1, 2 y 3 (vino blanco) y
11 en la cuba 4 de vino tinto. Resultó una media de 6,2 aislamientos por muestra. Los porcentajes
de aislamiento en cada una de las fases de muestreo se muestran en la Tabla 1.
Identificación de levaduras
Los resultados derivados del análisis taxonómico de las cepas de levaduras aisladas a lo largo
de la vinificación para las cuatro cubas, se encuentran expuestos en la Tabla 2. Se observa la presencia de 145 especies correspondientes a 7 géneros, 5 de los cuales son esporulados y 2 no esporulados (Candida y Kloeckera). Asimismo se puede verificar la evolución de cada una de las especies
aisladas a lo largo de las fases de fermentación, representado en porcentaje de aislamiento respecto
al total de cepas aisladas de la misma especie. Los valores X representan los valores promedio de
los tres tanques muestreados de vino blanco.
Dentro del grupo de levaduras no esporógenas, con metabolismo predominantemente oxidativo, destaca la presencia de 9 especies del género Candida (C apícola, C diversa, C glabrata, C guilliermondii, C lambica, C pulcherrima, C stellata, C solani y C valida). La mayoría de las especies
se aislaron en P. I. (período de inducción), mientras que en F.T. (fermentación tumultuosa) se aislaron C apícola, C glabrata, C pulcherrima y C valida. Finalmente, en EL. (fermentación lenta) se
recuperaron C. stellata, C. glabrata, C. pulcherrima, C valida y C guilliermondii. Dekkera
intermedia únicamente se aisló en una cuba de blanco en P.I. y F T ; Saccharomy codes ludwigii se
J. CANSADO, E. LONGO, D. AGRELO Y T. G. VILLA
83
TABLA 1
NUMERO DE CEPAS DE LEVADURAS Y PORCENTAJE DE AISLAMIENTO EN CADA FASE DE
MUESTREO DURANTE LA VINIFICACIÓN EN LAS CUATRO CUBAS DE FERMENTACIÓN
Fase de muestreo *
Cuba
1
2
3
4
Total
L'
2."
3."
4.'
5.'
6.'
9 (33,3)
8 (29,6)
5 (17,9)
3 (27,3)
5(18,5)
—
—
—
4(14,8)
8(29,6)
8(28,5)
5(45,4)
—
6(22,2)
—
—
9(33,3)
5(18,5)
7(25)
3(27,3)
—
—
8(28,5)
—
27
27
28
11
* Período de inducción (1/ y 2.^ fases), fermentación tumultuosa (3.^ y 4.'' fases), fermentación lenta (5.^ y 6.^
fases).
encontró en P.I. del tanque 3 para luego desaparecer. Kloeckera apiculata se recuperó en P.I. de todos los tanques excepto en el 3. Torulaspora delbrueckii presentó un aislamiento elevado en las tres
cubas de blanco y en todas las fases excepto en P.I. del tanque 2 (en el tanque 4 se aisló sólo en
F.T.). Por último, S. cerevisiae, la especie fermentiva más importante responsable del proceso de vinificación se aisló en la cuatro cubas estudiadas y en todas las fases, a excepción de P.I. de las cubas
3 y 4.
En cuanto a los resultados medios en las tres cubas de blanco, Candida, arrojó un porcentaje
medio de aislamiento variable según la especie de que se tratara. Así, destacan C glabrata y C. valida: la primera presentó un mayor porcentaje de aislamiento en EL., mientras que en P.I. y en F.T.
los valores fueron idénticos; la segunda mostró un porcentaje de aparición mayor en P.I., para disminuir en F.T. y volver a aumentar al final de la fermentación. K. apiculata y S, ludwigii solamente
se aislaron en PL, mientras que Debaryomyces hansenii sólo lo fue en EL. D. intermedia apareció
tanto en P.I. como F.T. T, delbrueckii se aisló en porcentaje idéntico en F.T. y EL., siendo los valores
algo inferiores en P.I. Por último, S, cerevisiae mantuvo un progresivo aumento a lo largo de la fermentación, pasando de un 16 % en P.I. a un 36 % en F.T., para terminar en un 48 % en EL. En la
cuba de tinto ha sido, sin embargo, más elevado el porcentaje de aislamiento en F.T. (66 %) que en
EL. (33 % ) . Es importante destacar que un 60 % de las cepas aisladas de esta especie presentaron
crecimiento positivo en medio con 50 % de D-glucosa.
Características de esporulación y homotalismo en las cepas de S. cerevisiae
En todas las cepas de S. cerevisiae vínicas aisladas a lo largo de las sucesivas fases de fermentación (28 cepas) se estudió su capacidad de esporulación, así como la viabilidad de las esporas; finalmente se evaluó si se trataba de cepas homo- o heterotálicas (Tabla 3). La mayoría de las cepas
mostraron una capacidad de esporulación igual o superior al 50 %, llegando en algunos casos a valores máximos, de 80 a 85 % (SMRl, SMR6, SMR19 y SMR21); también existen cepas con muy bajo índice de esporulación (1-20 %), como son SMR3, SMR13, SMR16, SMR25 y SMR27.
Un dato complementario al del porcentaje de esporulación lo constituye el número de ascosporas más frecuente que se encuentra en los cultivos esporulados. En la mayoría de las cepas existe
un predominio de aseas con dos y cuatro ascosporas; sin embargo, se detectaron algunas cepas que
presentaban esporulaciones atípicas, predominantemente con aseas de dos (SMR8, 25 y 27) y tres
ascosporas (SMR13).
LEVADURAS ASOCIADAS A PROCESOS DE FERMENTACIÓN.
84
TABLA 2
FRECUENCIAS DE AISLAMIENTO DE LEVADURAS EN DIFERENTES CUBAS Y
FASES DE FERMENTACIÓN
Especies
Saccharomyces cerevisiae
Torulaspora delbrueckii
Debaryomyces hansenii
Kloeckera apiculata
Saccharomycodes ludwigii
Dekkera intermedia
Candida apicola
Candida diversa
Candida glabrata
Candida guilliermondii
Candida lambica
Candida pulcherrima
Candida solani
Candida stellata
Candida valida
Fase de
muestreo *
PI
FT
FL
PI
FT
FL
PI
FT
FL
PI
FT
FL
PI
FT
FL
PI
FT
FL
PI
FT
FL
PI
FT
FL
PI
FT
FL
PI
FT
FL
PI
FT
FL
PI
FT
FL
PI
FT
FL
PI
FT
FL
PI
FT
FL
Cubal
Cuba 2
Cuba 3
y^ **
Cuba 4
9,1
27,3
63,6
33,3
33,3
33,3
42,4
28,8
28,8
—
50
50
16,0
36,0
48,0
25
37,5
37,5
—
57,1
42,9
33,3
33,3
33,3
—
—
—
—
_
—
100
100
100
100
100
100
—
—
—
—
—
—
—
—
100
100
~
—
—
—
50
50
—
100
—
—
100
—
—
—
—
50
50
—
—
100
—
—
50
50
—
—
50
50
—
100
100
—
—
—
25
25
50
50
50
50
100
—
66,7
33,3
—
100
50
50
—
—
—
50
100
100
100
I
—
100
100
100
—
—
—
—
—
—
—
50
50
100
—
—
—
100
—
—
—
—
—
—
—
—
—
—
—
—
100
33,3
33,3
33,3
100
57,1
14,3
28,6
—
—
—
~
—
—
100
50
—
—
—
50
* PI: período de inducción; FT: fermentación tumultuosa y FL: fermentación lenta.
** Valores media para las cubas 1,2 y 3 (vino blanco); cuba 4 (tinto).
100
85
J. CANSADO, E. LONGO, D. AGRELO Y T. G. VILLA
TABLA 3
CARACTERÍSTICAS DE ESPORULACIÓN, HOMOTALISMO Y FENOTIPO KILLER DE LAS
CEPAS VÍNICAS
DE
Cepa/muestreo *
SMRl/PI
SMR2/FT
SMR3/FT
SMR4/FT
SMR5/FL
SMR6/FL
SMR7/FL
SMR8/FL
SMR9/FL
SMRIO/FL
SMRll/FL
SMR12/PI
SMR13/PI
SMR14/PI
SMR15/FT
SMR16/FT
SMR17/FL
SMR18/FL
SMR19/FT
SMR20/FT
SMR21/FT
SMR22/FT
SMR23/FL
SMR24/FL
SMR25/FL
SMR26/FT
SMR27/FT
SMR28/FL
S. CEREVISIAE
Esporulación **
(%)
Ascosporas
por asea ***
80
65
20
35
60
85
30
53
70
50
25
75
1
50
60
20
30
50
80
50
80
55
50
50
15
75
10
58
2,4
2,4
2,4
2,4
2,4
4
2,4
2
2,4
2,4
1,2,4
2,4
3
2,4
4
4
2,4
4
2,4
2,4
2,4
2,3
2,4
4
2
2,4
2
4
Viabilidad
Ascosporas (%)
Homotalismo
64
68
25
100
75
90
100
100
81
86
42
87
+
+
+
+
+
+
+
+
+
+
+
+
—
100
50
56
82
+
+
+
+
—
50
68
55
69
96
75
+
+
+
+
+
+
—
93
+
—
100
+
Killer****
S
K2
S
K2
K2
s
S
S
K2
S
S
S
S
S
S
S
S
S
S
K2
K2
S
S
S
S
S
s
S
* PI: período de inducción; FT: fermentación tumultuosa, y FL: fermentación lenta.
** Sobre 200 células en cámara de Neubauer.
*** Número de esporas más abundante por asea.
**** K2 : fenotipo killer del tipo 2; S: fenotipo sensible.
Todos los cultivos de S. cerevisiae que presentaron índices de esporulación adecuados, así como aseas con los cuatro productos meióticos, fueron micromanipulados para estudiar las características de viabilidad de las ascosporas. El porcentaje de viabilidad se efectuó en todos los casos sobre
un mínimo de 24 ascosporas (6 tetradas) por cultivo. Los resultados obtenidos muestran valores de
viabilidad altos para la mayoría de las cepas, siendo en todos los casos mayores o iguales al 50 %,
salvo para las cepas SMR3 y SMRll, con valores del 25 y 42 %, respectivamente. Cinco cepas presentaron un 100 % de viabilidad de los productos meióticos: SMR4, 7,8,14 y 28.
Los cultivos de las esporas obtenidas por micromanipulación para cada cepa se conjugaron con
cepas heterotálicas a y a, no observándose en ningún caso la formación de zigotos. Por otra parte,
los cultivos procedentes de las esporas se sembraron en medio de esporulación, verificándose en la
práctica totalidad de los casos la formación de aseas. Estos nuevos cultivos esporulados fueron mi-
86
LEVADURAS ASOCIADAS A PROCESOS DE FERMENTACIÓN...
cromanipulados de nuevo, y los cultivos obtenidos inducidos a esporular, lo que ocurrió en la práctica totalidad de los casos.
Fenotipo killer en las cepas de S. cerevisiae
En las cepas de S. cerevisiae se realizó un test de actividad killer cuyos resultados se expresan
en la Tabla 3. En aquellas que no presentaron dicha actividad se estudió si se trataba de cepas sensibles o resistentes al carácter killer. Para ello se sembraron en lugar de la cepa sensible de colección,
y sobre ellas se inocularon cepas killer Ki y K2. En todos los casos se verificó la aparición de halos
de inhibición de crecimiento, por lo que se trató de cepas sensibles a la toxina killer. La cepas que
mostraron actividad killer fueron probadas para determinar qué tipo de carácter killer poseían. Los
resultados demostraron que se trataba de cepas killer K2. En cuanto a la distribución del fenotipo
killer a lo largo de la fermentación (Tabla 4), se observa que se aislaron en total 6 cepas killer K2
(SMR2,4,5, 9, 20 y 21), que representan un 21 % del total de S. cerevisiae aislados; de estas 6 cepas
no se aisló ninguna correspondiente al período de inducción, mientras que 4 corresponden a la fase
de fermentación tumultuosa y 2 a la fase lenta de fermentación. Por tanto, el porcentaje de aislamiento es significativamente más elevado en ET. que en EL. (un 36 % en la primera por un 15 %
en la segunda).
TABLA 4
DISTRIBUCIÓN DEL FENOTIPO KILLER DURANTE LA
FERMENTACIÓN
Muestreo
N. de cepas de
5. cerevisiae
N. de cepas
killer (%)
PI
FT
FL
4
11
13
0 (0)
4 (36)
2 (15)
TOTAL
28
6(51)
Discusión
La población de carácter fermentativo elevado está representada únicamente por S. cerevisiae.
Dentro de esta especie se han detectado hasta 10 grupos, que pueden diferenciarse en base a la asimilación de ciertos compuestos carbonados (L-lactato, melezitosa y succínico) y otras pruebas como crecimiento a 37° C y en presencia de glucosa al 50 %, siendo para este último carácter (importante en los procesos de fermentación), un porcentaje elevado de cepas las que muestran un resultado positivo. Otros géneros, como Torulaspora, Debaryomyces o Dekkera, presentaron un poder
alcohológeno y fermentativo débil (16). Esta distribución de especies parece estar relacionada con
una cierta lentitud de estas fermentaciones, ya observada por otros autores (9). K. apiculata aparece
únicamente en fases primarias de fermentación, como cabía esperar (18), desapareciendo en fases
posteriores debido a su baja resistencia al etanol. Destaca la considerable proporción de levaduras
del género Candida que presentan un carácter predominantemente oxidativo y productor de ácido
acético (1). Su presencia puede deberse por una parte a una escasa higiene en bodega, pues estas
especies son aisladas fácilmente entre la flora de bodega (6,16), y por otra al mal estado de la vendimia, dado el elevado índice de pluviosidaí* t.le la cosecha.
J. CANSADO, E. LONGO, D. AGRELO Y T. G. VILLA
87
Salvo casos aislados, la mayoría de la cepas aisladas esporularon apreciablemente, llegando en
algunas a valores del 80 %. De todas formas hay que indicar que los datos de esporulación se refieren a una temperatura de incubación de 30° C, y dado que se trata de un proceso termosensible,
probablemente los porcentajes observados mostrarían variaciones si la incubación se llevara a cabo
a otras temperaturas. Por otra parte, las esporas de una cepa vínica poseen una viabilidad que oscila
entre un 15 y 90 % (21), valores que se obtienen en el presente trabajo y que para algunas cepas llega a ser del 100 %. En cuanto a este aspecto, existen diferencias con respecto a las cepas de cervecería (2). Ello puede ser debido a la existencia en estas cepas de formas triploides, poliploides o
aneuploides, que se encuentran genéticamente desbalanceadas (5).
La práctica totalidad de los cultivos de esporas derivados de las levaduras aisladas en el presente estudio fueron capaces de esporular, pero no conjugaron con las cepas a y a de colección. Este
hecho sugirió que se trataba de cepas poliploides u homotálicas. Las cepas tetraploides son relativamente frecuentes en la naturaleza, pero no así las pentaploides o hexaploides. Para eliminar la posibiUdad de poliploidía, los cultivos esporulados derivados de las esporas fueron sometidos a análisis
de tetradas, y los clones derivados de éstas volvieron a esporular, con lo cual, de tratarse de cepas
poliploides, deberían ser al menos octaploides, por lo que cabe concluir que se trata de cepas homotálicas. El hecho de que la amplia mayoría de las cepas de levaduras vínicas sean homotálicas puede
significar que esta característica sirve como un mecanismo conservativo que contribuye a la estabilidad de la cepa y, por tanto, una característica deseable para ella (21).
En cuanto al fenotipo killer, aquellas cepas que resultaron killer-positivo, mostraron ser el tipo
2. Aunque la actividad killer manifiesta actividades máximas a valores de pH en torno a 4,5 (14,19),
se ha encontrado que en fermentación vínica a pH de mosto (3, 5) también se manifiesta, aunque en
menor medida, la actividad killer sobre cepas sensibles (19). Cabría esperar, por tanto, que a lo largo de la fermentación se fuese mostrando una predominancia de cepas killer sobre el resto de las
cepas, a menos que existiesen cepas killer resistentes (que no hemos encontrado). Sin embargo, no
hemos detectado un dominio acusado en la fermentación de cepas de S. cerevisiae de fenotipo killer. Además, los porcentajes significativamente más elevados de aislamiento de cepas killer en fases de fermentación tumultuosa, que en fermentación lenta pueden ser explicados en base a que la
producción de toxina se mantiene paralela al crecimiento durante la fase exponencial, comenzando
su degradación o inactivación al iniciarse la fase estacionaria de crecimiento (Bergillos, L., 1987. Tesis Doctoral, Universidad de Oviedo). De esta forma la menor actividad de la toxina coincidiría con
un aumento de poblaciones de cepas sensibles de S. cerevisiae.
Agradecimientos
El desarrollo del presente trabajo se financió a través de la Comisión Interministerial de Ciencia y Tecnología (Plan Nacional de Biotecnología), así como de la Excma. Diputación Provincial de
Orense.
Bibliografía
1. Amerine, M. A. and Kunkee, R. E. (1968). Microbiology of winemaking. Ann. Rev. Microb. 22,323-357.
2. Anderson, E. and Martin, P. A. (1975). The esporulation and mating of brewing yeasts. J. Inst. Brewing. 81,242-247.
3. Barnett, J. A., Payne, R. W. and Yarrow, D. (1983). Yeasts: Characteristics and Identification. Cambridge University
Press. Cambridge.
4. Buck, K. v., Lhoas, R, Border, D. J. and Saville, D. (1973). Virus particles in yeast. Biochem. Soc. Trans. 1,1141-1142.
5. Clayton, E. Howard, G. A. and Martin , P A. (1972). Yeast hybridisation. Proc. Am. Soc. Brewing Chemists. 72,78-81.
88
LEVADURAS ASOCIADAS A PROCESOS DE FERMENTACIÓN...
6. Cuinier, C. (1980). Origine des levures assurant l'élaboration d'un vin blanc de Touraine. Identification des espèces.
Conn. Vigne Vin. 14,111-126.
7. Fowell, R. R.; (1969). Sporulation and hybridization of yeasts. In: A. H. Rose and J. S. Harrison (eds.). The Yeasts.
Vol. 1, pp. 303-383. Academic Press. London.
8. Herskowitz, I. and Oshima, Y. (1981). Control of cell type in S. cerevisiae: Mating type and mating-type interconversion.
In: J. N. Strathem, E. W. Jones and J. R. Roach (eds.). The molecular biology of the yeast Saccharomyces cerevisiae,
pp. 181-210. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y
9. Iñigo, B. y Bravo, F. (1977). Estudio de mostos y vinos de Galicia. Agroq. Tecnol. Alim. 17,268-276.
10. Lodder, J. and Kreger-van Rij, N. J. W. (1952). The yeasts. A Taxonomic Study. North-Holland Publishing Company
Amsterdam.
11. McClary, D. G., Nulty, W. L. and Miller, G. R. (1959). Effect of potassium versus sodium in the esporulation of Saccharomyces. J. Bacteriol. 78,362-368.
12. McKover, M. and Bevan, E. A. (1963). The inheritance of a killer character in yeast S. cerevisiae. Proc. Int. Congress of
Genetics. XI1,202.
13. Naumova, G. I. and Naumov, T. I. (1973). Comparative genetics of yeasts. XIII Comparative Study of Killer Strains of
Saccharomyces from different collections. Genetika. 9,85-90.
14. Ouchi, K., Kawase, N., Nakano, S. and Akiyama, H. (1978). Stabilization of yeast Killer factor by glicerol. Agrie. Biol.
Chem.42,1-5.
15. Quecedo, C. R., Somavilla, J. E, Arroyo, V. e Iñigo, B. (1976). Agentes de fermentación de mosto de uva de la zona de
Galicia. Agroq. Tecnol. Alim. 16,123-130.
16. Ribereau-Gayon, J., Peynaud, E., Ribereau-Gayon, P. et Sudraud, P. (1975). Sciences et techniques du vin. Ed. Dunod.
Pans.
17. Ribereau-Gayon, P. (1985). New developments in wine microbiology. Am. J. Enol. Vitic. 36,1-9.
18. Sapis-Domerq, S. (1972). Comportament des levures apiculeés au cours de la vinification. Conn. Vigne Vin. 2,379-392.
19. Seki, T., Choi, E. and Ryu, D. (1985). Construction of killer wine yeast strain. Appl. Environ. Microbiol. 49,1211-1215.
20. Sherman, E, Fink, G. R. and Lawrence, C. W. (1979). Methods in yeast genetics. Cold Spring Harbor Laboratory, Cold
Spring Harbor. N. Y
21. Thornton, R. J. and Eschenbruch, R. (1976). Homothallism in wine yeasts. Antonie van Leeuwenhoek. 42,503-509.
22. Wickner, R. B. (1981). Killer systems in S. cerevisiae. In: J. N. Setrathern, E. W. Jones and J. R. Roach (eds.). The molecular biology of the yeast Saccharomyces cerevisiae, pp. 415-444. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y
MICROBIOLOGÍA SEM
5 (1989), 89-94
Ultraestructura del desarrollo intracelular del
bacteriófago 0C31
Ana Rodríguez, Carlos Hardisson y Juan E. Suárez *
Departamento de Biología Funcional. Área de Microbiología. Universidad de Oviedo. Julián Clavería, s/n.
Oviedo, Spain.
(Recibido mayo 20/julio 15, 1989)
Summary
The intracelullar development of the bacteriophage 0C31 in thermally induced cultures of the
lysogen Streptomyces coelicolor 01 changes remarkably its cell structure. At 10 min post-induction,
a big number of mesosomes are shown by the cells. At 30 min post-induction, the cytoplasm contains capsids which are still empty. At the end of the latent period mature virions are shown and immediately after, cell lysis occurs through the tip of germinative tubes. In old cultures (10 h or more)
no viral progeny is detected. However, when the amino acid glycine is added to the culture medium,
new virions are seen, but in smaller number than in germinating cultures. These results seem to
indicate that the lysis happens at the tip of the germinative tubes probably because this is an area
weakened by the preferential growth that takes place on it.
Key words: Bacteriophage, 0C31, ultrastructure, Streptomyces.
Resumen
El desarrollo intracelular del fago 0C31 en cultivos de la cepa lisogénica Streptomyces coelicolor 01 inducidos térmicamente, provoca cambios notables en la estructura celular. Dichos cambios
ya son evidentes 10 min después de la inducción, momento en el que las células muestran abundancia de mesosomas; a los 30 min son visibles en el citoplasma celular cápsidas virales vacías en su mayor parte; al final del período de latencia se observan viriones maduros; 60 min después de la inducción las células ya han empezado a lisarse, produciéndose la ruptura en el ápice de los tubos germinativos. En cultivos viejos (10 h o más), no se detecta progenie viral. Sin embargo, cuando el
lisógeno crece en presencia de glicina, la cual debilita las paredes celulares, se observa la aparición
de nuevos viriones, aunque en menor número que en cultivos germinantes, lo cual parece indicar
que, en estos últimos, la sahda de partículas fágicas se produce a través del ápice de los tubos germinativos por ser la zona más débil de la pared al producirse allí el crecimiento de forma preferencial.
Introducción
El estudio del desarrollo lítico del bacteriófago 0C31 de Streptomyces presenta la limitación
de su baja capacidad infectiva, lo cual determina que los ciclos líticos sean asincrónicos (9). Para su(*) A quien se dirigirá la correspondencia.
90
ULTRAESTRUCTURA DEL DESAROLLO INTRACELULAR...
perar este problema hemos construido un lisógeno termoinductible. Este permite la iniciación del
desarrollo fágico en todas las células simultáneamente cuando se exponen a cultivos de esporas germinantes a 40° C durante 5 min (12). Los períodos de eclipse y latencia en estas condiciones son de
20 y 45 min respectivamente, habiéndose observado que únicamente se produce progenie viral
cuando el cultivo es joven. No obstante, en cultivos viejos se produce la muerte celular como consecuencia de la inducción térmica. El desarrollo viral va a alterar profundamente el modelo de biosíntesis macromolecular en las células. Así, la síntesis de ARN celular se inhibe casi inmediatamente
después de la inducción, existiendo datos que sugieren la modificación de las ARN polimerasas,
que de este modo realizarán la transcripción preferencial del ADN viral, cuya replicación se inicia a
los 10 min post-inducción (12,13). Por otro lado se ha realizado un mapa de transcripción viral mediante la detección y localización en su genoma de las secuencias complementarias a 16 especies de
ARN, de las cuales 12 son de transcripción temprana y vida media muy corta, siendo solamente detectados hasta 10-15 min post-inducción, y los 4 restantes son de transcripción tardía. Entre estos últimos se ha detectado un ARN policistrónico de unas 13 Kb (14).
En el presente artículo presentamos resultados que muestran los cambios morfológicos que sufren las células de la cepa lisogénica tras la inducción del ciclo de desarrollo del fago 0C31, así como datos que permiten explicar la ausencia de progenie viral exocelular en cultivos viejos.
Materiales y métodos
Organismos y métodos microbiológicos
El estudio del desarrollo intracelular del fago 0C31 se llevó a cabo en cultivos inducidos de
Streptomyces coelicolor 01, cepa lisogénica para el mutante termoinducible 0C31 cts (10), en medio
GAE fortificado (12). La enumeración de la progenie viral se realizó en medio agar común suplementado con MgS04 10 mM y Ca (N03)2 8 mM, utilizando como cepa indicadora Streptomyces lividans 66, cuyas esporas se incorporaron en la capa superior de agar semisólido. Las condiciones de
esporulación, obtención de esporas, almacenamiento, etc., han sido pubhcadas previamente (12).
Microscopía electrónica
Las muestras fueron tratadas según se ha descrito (6, 7), con la única excepción de que los cortes ultrafinos se contrastaron mediante una solución de acetato de uranilo al 2 % en etanol al 50 %,
durante 5 min, lavándose a continuación con agua destilada. Seguidamente se realizó un tratamiento con citrato de plomo en las condiciones descritas con anterioridad (11). La observación se realizó
en un microscopio Zeiss EM 109 a 50 Kv, y se utilizó película Kodak grano fino 5 302 de 35 mm para la obtención de microfotografías.
Resultados y discusión
A las 7 horas de incubación a 28° C y en agitación en medio GAE fortificado, los cultivos iniciados con esporas durmientes de S. coelicolor 01 están compuestos por hifas de 5-8 |im de largo, en
uno de cuyos extremos es visible aún la espora a partir de la cual se originaron.
Su estructura es la característica de cultivos de actinomicetos creciendo activamente (6, 8).
Así, es observable una pared de apariencia uniforme que rodea la membrana plasmática trilamelar,
y un citoplasma en el que los ribosomas son abundantes y el ADN aparece muy laxo, indicando que
existe una transcripción muy activa (Fig. 1 A). La inducción térmica de los cultivos provoca cam-
A. RODRIGUEZ, C. HARDISSON Y J. E. SUAREZ
91
Fig. 1. Ultraestructura del desarrollo intracelular del fago 0C31: A) Sección transversal de célula de un cultivo de Streptomyces coelicolor 01 no inducido, a las 7 h de germinación, p: pared celular; m: membrana celular. B) Sección transversal de célula
de un cultivo de S. coelicolor 01 10 min después de la inducción. La célula muestra formaciones membranosas de tipo mesosómico (ms) muy desarrolladas. C) Sección transversal de célula de S. coelicolor 01 30 min después de la inducción. Se distingue
perfectamente la pared (p) y la membrana (m) celulares, y un mesosoma (ms) muy desarrollado. En este momento se hacen
visibles las cabezas fágicas (c) vacías, poco densas a los electrones. La barra equivale a 200 nm.
bios importantes en la estructura de las células: a los 10 min post-inducción (Fig. 1 B), las células presentan multitud de mesosomas, que son también evidentes a los 30 min post-inducción (Fig. 1 C),
momento en el cual ya se observan en el citoplasma cápsidas virales que en su mayoría están aún
vacías, como se deduce de su baja densidad electrónica. A los 45 min post-inducción (Fig. 2 A), las
cápsidas aparecen más densas a los electrones, indicando que ya se ha producido el ensamblaje de
los viriones, y a los 60 min, prácticamente todo el cultivo aparece lisado (Fig. 2 B), siendo destacable el que la ruptura de las células ocurra a nivel del ápice de las hifas. El hecho de que en los organismos filamentosos el crecimiento de las hifas sea preferentemente apical, tanto en el caso de hongos (1, 4) como en Streptomyces (3), permite postular que dicho ápice es una zona especialmente
débil de la pared celular, por lo que la lisis de las células ocurrirá preferencialmente en ese punto
por efecto de la endolisina del fago.
92
ULTRAESTRUCTURA DEL DESAROLLO INTRACELULAR.
Fig. 2. Ultraestructura del desarrollo intracelular del fago 0C31. A) Sección longitudinal de un tubo germinativo de S. coelicolor 01 45 min después de la inducción. Aparecen cabezas fágicas (c) maduras, densas a los electrones, en las que ha penetrado
del DNA fágico. B) Sección longitudinal de célula de 5. coelicolor 01 60 min después de la inducción. En la fase de lisis, las células comienzan a Usarse por la zona apical (a) del tubo germinativo. La barra equivale a 200 nm.
Por otro lado, este último dato permite plantear la hipótesis de que sería posible que la no aparición de progenie fágica en cultivos inducidos después de 10 h de incubación, fuese debida, al menos en parte, a la incapacidad de los enzimas líticos virales para romper las paredes del micelio viejo que presenta su peptidoglicano modificado, ya que no es susceptible de digestión con lisozima
(2). Para aclarar esta cuestión se incubaron esporas de S. coelicolor 01 en presencia de glicina al
1 % y en ausencia del aminoácido, dada la capacidad del mismo para debilitar las paredes de las células que crecen en su presencia (5). Alternativamente, los cultivos fueron sometidos a tratamiento
con lisozima (10 mg/ml cf) durante 2 h a 28° C. Los cultivos se incubaron hasta alcanzar la fase estacionaria (15 h), momento en el que se sometieron a inducción térmica, incubándose entonces 2 h
más para permitir la producción de progenie viral. Transcurrido este período se extrajo una alícuota
para determinar la concentración de viriones, y al resto se añadió lisozima, prosiguiendo la incubación 2 h más. Los resultados (Tabla 1) indican que la progenie viral obtenida era significativamente
mayor en los cultivos incubados en presencia de aminoácido, mientras que la presencia de lisozima
A. R O D R I G U E Z , C H A R D I S S O N Y J. E. S U A R E Z
93
TABLA 1
R E N D I M I E N T O (1) EN P R O G E N I E V I R A L D E CULTIVOS D E STREPTOMYCES
COELICOLOR
I N C U B A D O S E N P R E S E N C I A O A U S E N C I A D E GLICINA, E I N D U C I D O S T É R M I C A M E N T E
Medio de cultivo
G A E f (2)
GAEf
GAEf+Gly(l%)
G A E f + lisozima (3)
GAEf + Gly (1 % ) + lisozima
Momento de la inducción (h)
Rendimiento
7
15
15
15
15
500
5,2
20
5
18,29
01,
(1) Rendimiento: Relación entre el número de pfu/ml obtenido en cultivos inducidos con respecto al obtenido
en cultivos controles.
(2) GAEf: Al medio fortificado se le añadieron 5 gr/1 de extracto de levadura.
(3) La lisozima (10 mg/ml cf) se añadió a cultivos inducidos e incubados durante 2 h para permitir la formación
de progenie viral, incubándolos durante 2 h más a 28° C.
no variaba el resultado. Además, en el microscopio de contraste de fases los cultivos crecidos en
presencia de glicina e inducidos, aparecían lisados mientras que el resto no lo estaban. Sin embargo,
el rendimiento en pfu/ml es menor que el obtenido en cultivos germinantes, en los que llega a ser
500 veces superior al mostrado por los cultivos controles (12) (Tabla 1). Es, pues, evidente que el
estado fisiológico de las células influye en el tamaño de la progenie viral. Esto se confirma porque
no aparecen cápsidas virales en secciones de células en fase estacionaria (dato no mostrado), probablemente por la escasez de las mismas en estas condiciones junto con la imposibilidad física de los
viriones que se producen por inducción espontánea o térmica para salir de las células. Dicha imposibilidad se debe posiblemente a la incapacidad de las endolisinas virales para hidrolizar la pared
celular de los cultivos viejos de Streptomyces.
Bibliografía
1. Bartnicki-García, S. and Lippman, E. (1969). Fungal morphogenesis: cell wall construction in Mucor rouxii. Science 165,
302-304.
2. Braña, A. R, Manzanal, M. B. and Hardisson, C. (1981). Cytochemical and enzymatic characterization of the sporulation
septum of Streptomyces antibioticus. Can. J. Microbiol. 27,1060-1065.
3. Braña, A. E, Manzanal, M. B. and Hardisson, C. (1982). Mode of action of cell wall growth of Streptomyces antibioticus.
FEMS Microbiol. Lett. 13,231-235.
4. Gooday, G. W. (1971). An autoradiographic study of hyphal growth of some fungy. J. Gen. Microbiol. 67,125-133.
5. Hammes, W., Schleifer, K. H. and Kandler, O. (1973). Mode of action of glycine on the biosynthesis of peptidoglycan. J.
Bacteriol. 116,1029-1053.
6. Hardisson, C. and Manzanal, M. B. (1976). Ultrastructural studies of sporulation in Streptomyces. J. Bacteriol. 127,14431454.
7. Hardisson, C , Manzanal, M. B., Salas, J. A. and Suárez, J. E. (1978). Fine structure, physiology and biochemistry of arthrospore germination in Streptomyces antibioticus. J. Gen Microbiol. 105, 203-214.
8. Hardisson, C. and Suárez, J. E. (1979). Fine structure of spore formation in Micromonospore chalcea. J. Gen. Microbiol.
110,233-237.
9. Lomovskaya, N. D., Chater, K. F. and Mkrturmian, N. M. (1980). Genetics and molecular biology of Streptomyces bacteriophages. Microbiol. Rev. 44,206-229.
10. Novikova, N. L., Kapitonova, O. N. and Lomovskaya, N. D. (1973). Thermal prophage induction in germinating spores
oí Streptomyces coelicolor A3(2) (0C31 ct 1). Microbiologiya 45, 713-718.
11. Reynolds, E. S. (1963). The use of lead citrate at high pH as an electron opaque stain in electron mycroscopy. J. Cell.
Biol. 17,208-212.
94
ULTRAESTRUCTURA DEL DESAROLLO INTRACELULAR...
12. Rodríguez, A., Caso, J. L., Hardisson, C. and Suárez, J. E. (1986). Characteristics of the developmental cycle of actinophage 0C31. J. Gen. Microbiol. 132,1695-1701.
13. Rodríguez, A., Hardisson, C. and Suárez, J. E. (1988). The effect of rifampicin on the development of the Streptomyces
bacteriophage 0C31. Microbiología SEM 4,47-53.
14. Rodríguez, A., Chater, K. E, Hardisson, C. and Suárez, J. E. (1989). Transcription map of bacteriophage 0C31. J. Virol,
(enviada).
M I C R O B I O L O G Í A SEM
5 (1989), 95-103
Prevalence of Salmonella serotypes isolated in Spain
from human and non human sources (1983-1987)
M. A. Echeita * and M. A. Usera
Laboratorio de Referenda de Salmonella. Servicio de Bacteriología. Centro Nacional de Microbiología. Majadahonda. Madrid
(España)
(Received May 3/July 25,1989)
Summary
Salmonella serotypes over a five year period were studied in order to know their prevalence in
Spain. The Salmonella Reference Centre received a total of 11 fill strains from 1983-1987. The majority (16,133) were of human origin and only 1,479 strains were isolated from non-human sources.
The sero typing yielded 100 different serotypes. Salmonella entérica serotype Enteritidis (8) being
the commonest in both groups, 61.18% of human origin and 31.91 % of non-human origin. Salmonella entérica serotype Typhimurium the commonest serotype in many countries, occupies second
place in our results with the following percentages 11.87 % and 9.67 % respectively. Among the
strains of human origin Salmonella entérica serotype Typhi occupies fourth place (3.24 %). This is
very low compared with the high number of clinically diagnosed typhoid fever cases declared in the
country: over 5,000 cases per year.
Key words: Salmonella, serovars, prevalence.
Resumen
Con objeto de conocer la prevalencia de los serotipos de Salmonella en España, se han estudiado en el Centro Nacional de Referencia de Salmonella las cepas recibidas durante 5 años (19831987). De las 17.612 cepas recibidas la mayor parte (16.133) fueron de origen humano y solamente
1.479 se aislaron de otras fuentes. El serotipado presentó 100 serotipos diferentes, siendo Salmonella entérica, serotipo Enteritidis (8) el más frecuente en ambos grupos, 61,18 % en las cepas de origen humano y 31,91 % en las de otros orígenes. Salmonella entérica, serotipo Typhimurium, que es
en otras comunidades el serotipo más frecuente, en nuestros resultados ocupa el segundo lugar con
un 11,87 % y 9,67 % respectivamente. En las cepas de origen humano el cuarto lugar corresponde a
Salmonella entérica, serotipo Typhi con un porcentaje muy pequeño (3,24 %) comparado con el alto número de casos de fiebre tifoidea declarados por diagnóstico clínico en España (más de 5.000
casos por año).
Introduction
The Salmonella foodborne outbreaks represent a very important problem world wide, not only
for its incidence in human gastroenteritis (in Spain it is the principal etiological agent of gastroente(*) Corresponding author.
96
PREVALENCE OF SALMONELLA SEROTYPES...
ritis) (5) but also due to its importance in other economic sectors, such as the processed food industry, the tourist trade, etc. Serotyping is the most useful epidemiological marker for Salmonella,
differentiating over 2,000 serotypes according to the Kauffmann-White Scheme (9).
The Salmonella laboratory of the National Centre for Microbiology of Majadahonda, Madrid,
has received Salmonella strains for serotyping since 1979, acting as the National Salmonella Reference Centre for Spain since 1984. The serotyping results are published annually in the «Boletin
Epidemiológico Semanal» (1) and in the «Boletin Microbiológico Semanal» (4).
In this study we examined 17,612 Salmonella strains submitted to the national Reference Centre from many diagnostic laboratories in Spain between 1983-1987 in order to know the serotype
prevalence, its evolution throughout this period, its source of isolation and the seasonal, sex and age
distribution of the isolates.
Materials and methods
Salmonella strains were isolated and identified by conventional methods in hospital and public
health laboratories situated in different parts of the country, mainly from human sources (faeces,
blood, etc.) and also from food (egg products, poultry, meat, etc.) environmental sources (river and
sea water, waste water, etc.) and from sick animals (sheep, chickens, etc.).
On receipt at the Reference Centre, each culture was replated onto McConkey agar for a purity
chek, before studying a set of biochemical characteristics, to confirm the presence of Salmonella (6).
The anigenic formula was established by slide agglutination and microagglutination (11) with
both polyvalent and specific sera. Swarm agar plates incorporating appropriate antisera for the selection of alternative flagellar phases were used. Additional biochemical tests were set up if indicated. Strains that gave rise to diagnostic problems were sent to the World Health Organization
(WHO) Collaborating Centre (Institut Pasteur. Paris) for serotyping. Serological reactions were
studied using sera prepared in our laboratory according to the Guidelines for the preparation of
Salmonella antisera (10).
Results
The National Salmonella Reference Centre for Spain received and confirmed a total of 17,612
Salmonella strains between 1983-1987. They were classified according to isolate sources into two
groups:
I. Strains of human origin (16,133).
II. Strains of non-human origin (1,479).
The number of strains received for serotyping increased annually (Table 1). The isolates pertaining to serogroups B, C and D represent 93.82 % (Table 2). We found 100 different serotypes whose isolate numbers are presented in Tables 3 and 4, grouped by serogroups. Serotypes whose
percentage is over 1 are represented in Tables 5 and 6. The more frecuently found serotypes in both
groups are similar, but there are significant percentage differences. The particular evolution of Enteritidis, Typhimurium and Virchow serotypes is shown in Figures 1 and 2 respectively.
Group I
The seasonal distribution shows an increasing number of isolates during the summer and at the
beginning of the autumn as was expected.
M. A. ECHEITA AND M. A. USERA
97
TABLE 1
ANNUAL DISTRIBUTION OF STRAINS RECEIVED 1983-1987
Strains of human origin
Strains of non-human origin
1983
1984
2,343
172
2,548
84
1985
3,126
170
1986
3,788
396
1987
4,328
657
Total
16,133
1,479
TABLE 2
SEROGROÜP DISTRIBUTION OF THE TOTAL NUMBER OF
STRAINS RECEIVED 1983-1987
Serogroup
A
B
C
C-1
C-2
C-3
D D-1
E E-1
E-4
F
G
I
M
Autoaglutinable
Others
TOTAL
Number of strains
Frecuency (%)
2
2,864
1,849
586
1
11,222
127
43
1
16
2
1
441
457
0.01
16.26
10.50
3.33
0.01
63.72
0.72
0.24
0.01
0.09
0.01
0.01
2.50
2.59
17,612
100.00
Faeces are the principal isolation source for strains (84.6 %) except for Salmonella ser. Typhi
whose principal source is blood (58.3 % ) . There is no significant difference between sexes, and the
age distribution shows a higher number of isolates in the group under 4 years. In the case of Salmonella ser. Typhi the adult group is more often implicated.
Group II
The principal strain isolation sources are food: poultry (30.5 %) and eggs (16.8 %) and of environmental origin: waste water (67.6 % ) . In 1987 the strains received from this group increased considerably: from 396 in 1986 to 657 in 1987.
Discussion
The annually increasing number of Salmonella strains serotyped by the National Salmonella
Reference Centre for Spain, allows us to improve our knowledge regarding Salmonella prevalence
in the country. Salmonella strains pertaining to serogroups B, C and D represent over 90 % of the
isolates, this allows us to suggest to Clinical and Public Health Laboratories to use a small set of
antisera groups to confirm biochemical identification.
98
PREVALENCE OF SALMONELLA
SEROTYPES.
TABLE 3
SEROTYPE DISTRIBUTION OF HUMAN ORIGIN STRAINS
Serogroup
A
B
C
C-1
C-2
Serotype
Paratyphi A
Agona
Azteca
Bochum
Bradford
Brandenburg
Bredeney
Canada
Coeln
Derby
Fyris
Hato
Heidelberg
Hessarek
Java
Lagos
Limete
Paratyphi B
Remo
Saintpaul
Sofia (II)
Schwarzengrund
Thyphimurium
Group B
Bareilly
Braenderup
Brazzaville
Colindale
GaHema
Hartford
Infantis
Isangi
Livingstone
Mbandaka
Mikawasima
Montevideo
Ohio
Oranienburg
Postdam
Richmond
Thompson
Virchow
Group C-1
Blockley
Bonaerensis
Bovismorbificans
Cremieu
Goldcoast
Hadar
Litchfield
Loanda
Manchester
Manhattan
1983
1984
1985
1986
1987
—
—
—
—
Ï
—
—
—
—
—
Ï
5
1
7
23
—
—
—
14
1
63
—
2
1
—
4
—
—
14
1
—
—
8
31
—
1
—
8
—
45
—
—
—
1
26
3
4
6
—
—
1
3
52
1
1
1
1
—
—
—
—
—
—
—
—
—
—
10
2
—
—
—
—
—
—
—
—
1
105
—
—
—
—
—
—
1
—
94
—
—
3
4
15
127
1
1
1
3
15
78
1
357
1
4
3
52
3
12
72
—
—
—
—
—
—
—
—
—
—
—
—
—
—
11
12
63
4
2
—
2
—
—
3
1
81
53
4
8
10
—
1
1
—
1
—
—
—
—
—
1
—
—
_
—
—
—
—
20
1
2
1
1
3
—
—
1
1
—
—
—
4
—
42
26
—
—
—
3
1
504
348
21
41
13
—
—
—
99
22
1
7
1
14
15
—
—
—
—
19
1
4
1
3
1
344
17
1
—
1
—
2
49
1
3
—
70 '
5
65
—
—
1
1
159
2
40
10
8
1
11
363
14
1
—
39
—
—
—
9
4
61
—
—
—
__
278
11
47
—
—
—
—
—
—
—
—
1
2
5
14
2
6
1
13
13
2
Total
^2
33
1
5
2
81
173
1
3
2
24
1
157
2
8
2
2
42
3
18
17
1
1,916
31
1
9
1
1
5
3
339
1
3
4
89
51
403
2
1
1
13
629
13
225
1
22
4
42
52
6
1
1
3
M. A. ECHEITA AND M. A. USERA
99
TABLE 3
(CONTINUED)
Serogroup
C-3
D D-1
E E-1
E-4
G
I
TOTAL
Serotype
Muenchen
Newport
Stourbridge
Takoradi
Group C-2
Hindmarsh
Ententidis
Goettingen
Kapemba
Mendoza
Panama
Typhi
Group D-1
Amsterdam
Anatum
Binza
Birmingham
Folkensee
Give
London
Meleagridis
Newbrunswick
Newington
Orion
Tournai
Vejle
Group E-1
Fareham
Fulda
Senftenberg
Tilburg
Westhampton
Group E-4
Havana
Ordonez
Poona
Putten
Worthington
Group G
Weston
Arizonae
Diarizonae
Autoaglutinable
Monophasic
Non motil
Sp
1983
1984
4
15
1
—
—
—
1,021
—
29
1
28
89
—
—
2
—
—
—
2
—
—
—
—
5
—
1
—
—
—
3
—
—
—
—
2
—
—
__
—
—
—
—
61
95
11
-—
4
7
2,343
1
—
—
1,382
—
11
—
28
77
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
35
62
7
77
1985
—
14
—
2
—
—
2,016
—
3
—
14
109
-_
1
1
3
—
1
2
3
—
1
—
—
—
—
—
—
1
—
1
—
—
—
—
—
—
—
—
—
—
—
81
—
—
102
1986
13
22
—
—
2
1
2,571
2
3
—
29
115
13
—
6
—
—
—
—
4
1
3
2
—
1
—
—
1
—
1
3
—
2
—
—
1
1
4
—
1
—
—
101
—
—
7
1987
25
26
—
—
1
—
2,976
1
6
—
34
132
6
—
11
—
1
—
3
16
—
2
—
—
—
—
3
—
—
3
6
1
—
1
—
—
—
—
2
—
1
1
83
—
—
12
Total
46
84
1
3
3
1
9,966
3
52
1
133
522
19
1
20
3
1
1
7
23
1
6
2
5
1
1
3
1
1
7
10
1
2
1
2
1
1
4
2
1
1
1
361
157
18
198
2,548
3,126
3,790
4,328
16,133
—
In many countries Salmonella ser. lyphimurium is the commonest serotype (2) (7), but our results here suggest that since 1982 the first place is occupied by Salmonella ser. Enteritidis^ (61.18 %
of the human origin strains) and this percentage is increasing annually. In strains of non human origin, although Salmonella ser. Enteritidis is also the commonest serotype (31.91 % ) , its frequency is
100
PREVALENCE OF SALMONELLA
SEROTYPES.
TABLE 4
SEROTYPE DISTRIBUTION OF NON-HUMAN ORIGIN STRAINS
Serogroup
B
C
C-1
C-2
D
E
D-1
E-1
Serotype
Abortusovis
Agona
Brandenburg
Bredeney
Derby
Fyris
Heidelberg
Java
Paratyphi B
Reading
Saintpaul
Salinatis
Sofia (II)
Typhimurium
Group B
Braenderup
Infantis
Livingstone
Mbandaka
Mikawasima
Montevideo
Ohio
Oranienburg
Othmarschen
Rissen
Thompson
Virchow
Group C-1
Blockiey
Bovismorbificans
Cleveland
Goldcoast
Hadar
Kottbus
Lichtfield
Manchester
Manhattan
Muenchen
Newport
Group C-2
Enteritidis
Goettingen
Jamaica
Kapemba
Panama
Group D
Anatum
Drypool
Give
London
Meleagridis
Newbrunswick
1983
1984
1986
2
4
9
10
2
1987
15
14
9
20
2
3
1
1
2
2
2
1
30
4
31
12
1
20
4
4
2
2
4
1
1
20
53
6
1
9
1985
5
—
—
6
1
6
12
5
1
4
1
1
1
4
3
—
—
22
7
8
—
6
1
—
—
—
—
1
15
2
2
5
8
1
4
8
—
—
—
—
—
1
1
1
4
2
1
34
5
6
5
—
—
—
—
—
—
—
—
—
—
—
—
3
9
1
—
1
9
7
3
13
29
3
1
—
—
1
9
3
1
—
4
1
1
1
1
—
—
—
—
—
1
—
1
1
24
—
—
—
—
1
68
4
13
—
10
1
—
—
—
5
4
21
71
171
—
—
—
1
25
5
—
1
2
—
—
—
—
—
—
3
—
1
—
-
2
1
2
1
1
2
1
5
185
2
1
9
2
18
1
1
13
1
Total
22
25
18
53
4
2
11
3
14
4
5
1
25
143
18
3
52
5
7
4
25
62
3
1
2
2
104
10
33
8
1
12
6
1
10
1
2
6
5
6
475
2
1
27
18
3
20
1
2
18
2
2
M. A. ECHEITA AND M. A. USERA
101
TABLE 4
(CONTINUED)
Serogroup
E-4
F
G
I
M
Serotype
Newington
Orion
Group E-1
Llandof
Senftenberg
Tilburg
Group E-4
Abaetetuba
Atlanta
Havana
Worthington
Welikade
Umbilo
Autoaglutinable
Monophasic
Non motil
Sp.
TOTAL
1983
—
—
—
—
—
—
—
—
1
—
—
—
—
5
13
4
1
1984
—
—
—
—
—
—
—
—
—
—
—
—
—
3
2
1
16
172
84
—
—
1986
2
—
—
—
2
—
1
—
—
1
—
1
1
—
—
10
24
—
—
4
1987
2
—
1
—
4
13
—
—
—
2
—
—
—
41
—
—
27
170
396
657
1985
—
—
—
—
—
—
—
Total
4
1
1
1
6
13
1
1
1
3
1
1
1
1,479
80
15
5
58
TABLE 5
MOST FREQUENTLY FOUND SEROTYPES OF HUMAN
ORIGIN (1983-1987)
Serotype
Enteritidis
Typhimurium
Virchow
Typhi
Ohio
Infantis
Blockley
Bredeney
Others
TOTAL
imber of strains
9,966
1,916
629
522
403
339
225
173
1,960
16,133
Frequency (%)
61.18
11.87
3.90
3.24
2.50
2.10
1.39
1.07
12.75
100.00
lower than among the strains of human origin. We can also appreciate a greater serotype dispersion
within this group although our experience is still limited. Compared with the clinically diagnosed
typhoid fever cases declared annually in Spain (3), mean 5,000, we have received very few Salmonella ser. Typhi strains (mean 100).
Salmonella ser. Mikawasima an uncommon serotype in Spain has suffered a considerable increase during 1986 due to an outbreak in Catalonia, that affected a large number of people. Finally we
want to sign the progressive increase of Salmonella ser. Virchow, and the slight decrease of Salmonella ser. Infantis during the period studied.
Acknowledgments
We thank the WHO Collaborating Centre for Reference and Research on Salmonella at the
Institut Pasteur (Paris), France, for serotyping problem strains.
We thank Rafaela Gutiérrez for expert technical assistance.
102
PREVALENCE OF SALMONELLA SEROTYPES...
TABLEÓ
MOST FRECUENTLY FOUND SEROTYPES OF HUMAN
ORIGIN (1983-1987)
Serotype
Ententidis
Typhimunum
Virchow
Ohio
Bredeney
Infantis
Blockiey
Kapemba
Agona
Montevideo
Sofia (II)
Abortusovis
Anatum
Brandenburg
London
Panama
Others
TOTAL
Total number of strains
472
143
104
62
53
52
33
27
25
25
25
22
20
18
18
18
362
Frequency (%)
1,479
31.91
9.67
7.07
4.22
3.60
3.53
2.24
1.83
1.70
1.70
1.70
1.49
1.38
1.22
1.22
1.22
24.30
100.00
78.9
•/.
I
S
0
L
A
T
E
S
•Uiï^chow iTyphirturlEnterit.
VEARS
Fig. 1. Evolution of serotypes Ententidis. Typhimunum and Virchow from human origin strains.
M. A. ECHEITA AND M. A. USERA
103
50.0
4e. e
30. ek
I
S
0
L
A
T
£
S
20.9^
10.0
0.
•Uirchos STyphiMuriEnterit.
VEARS
Fig. 2. Evolution of serotypes Enteritidis, Typhimurium and Virchow from non-human origin strains.
References
Editorial (1984). Tipificación de Salmonellas. 1983. Boletín Epidemiológico Semanal, Ministerio de Sanidad y Consumo,
Spain 1654,265-267.
Editorial (1985). Sahnonelosis humanas USA 1983. Boletín Microbiológico Semanal, Ministerio de Sanidad y Consumo,
Spain 3,1-8.
Editorial (1986). Estado de las enfermedades de declaración obügatoria. Boletín Epidemiológico Semanal, Ministerio de
Sanidad y Consumo, Spain 1766,383.
Editorial (1988). Análisis de los resultados de las cepas de Salmonella recibidas para su estudio en el Laboratorio de Enterobacterias del Servicio de Bacteriología del CNMVIS durante el año 1987. Boletín Microbiológico Semanal. Ministerio de Sanidad y Consumo, Spain 42-43,1-5.
Editorial (1988). Brotes de infecciones e intoxicaciones de origen alimentario. España, 1987. Boletín Epidemiológico Semanal, Ministerio de Sanidad y Consumo, Spain 1806,85-86 and 1807,97-98.
Ewing, W. H. (1986). Edwards and Ewing's Identification of Enterobacteriaceae, 4th edition. Elsevier Science Publishing,
New York.
Le Minor, L., Le Minor, S. and Grimont, P. A. D. (1985). Quatriennal report of the French National Salmonella Centre
on origin and distribution among serotypes of the strains isolated in continental French during years 1980 to 1983. Revue
Epidemiologique et Santé Publique 33, 13-23.
Le Minor, L. and Popoff, M. Y. (1987). Designation of Salmonella entérica sp. nov. nom. rev., as the type and only species
of the genus Salmonella. International Journal of Systematic Bacteriology 37,465-468.
Le Minor, L. and Popoff, M. Y (1988). Antigenic formulas of the Salmonella serovars 5¡th revision. WHO Collaborating
Centre for Reference and Research on Salmonella. Institut Pasteur Paris, France.
10. Le Minor, L., Bockemiilhl, J., Aleksic, S. and Popoff, M. Y (1989). Guidelines for the preparation of Salmonella antisera,
4th revision. WHO Collaborating Centre for Reference and Research on Salmonella, Institut Pasteur, Paris. France.
11. Shipp, C. R. and Rowe, B. (1980). A mechanised microtechnique for Salmonella serotyp»ing. Journal of clinical Pathology,
33,595-597.
MICROBIOLOGÍA SEM
5 (1989), 105-122
Selected characteristics of several strains of
Lactobacillus plantarum
Baltasar Mayo, Carlos Hardisson and Alfredo F. Braña*
Laboratorio de Microbiología. Departamento de Biología Funcional. Facultad de Medicina. Universidad de Oviedo.
33006 Oviedo, Spain.
(Received June 27/September 6,1989)
Summary
Several relevant characteristics have been studied in nine Lactobacillus plantarum strains isolated from Cabrales cheese. They fermented lactose efficiently and possessed both IB-galactosidase
and 6-phosphogalactosidase activities, but no significant proteolytic activity was detected. Antimicrobial susceptibility tests revealed no high level resistance to any of the fourteen compounds tested. All the strains had a high content of extrachromosomal DNA of unknown function. A recombinant plasmid comprising pUC19 from Escherichia coli and a small cryptic plasmid from L.plantarum has been constructed and may be used to develop a shuttle vector for these species.
Key words: Lactobacillus plantarum, Cabrales cheese, lactic acid bacteria.
Resumen
Se han estudiado varias características relevantes en 9 cepas de Lactobacillus plantarum aisladas de queso de Cabrales. Todas ellas fermentaron eficientemente la lactosa y presentaron actividades 6-galactosidasa y 6-fosfogalactosidasa, pero no se encontró actividad proteolítica significativa.
Los tests de susceptibilidad frente a 14 agentes antimicrobianos no revelaron resistencia a altos niveles de estos compuestos. Todas las cepas mostraron un alto contenido de DNA extracromosómico de función desconocida. Se ha construido un plásmido recombinante que comprende pUC19 de
Escherichia coli y un pequeño plásmido críptico de L. plantarum. Este nuevo plásmido podría ser
de utilidad en el desarrollo de un vector lanzadera para ambas especies.
Introduction
In recent years, there has been a growing interest in the genetics and phisiology of lactic acid
bacteria that are used in a variety of fermentations of great economical importance. The research in
this field may lead to the obtention of strains that would improve current processes or facilitate the
development of new ones. Most studies, however, have dealt with species of genus Lactococcus
(formerly lactic stroptococci) and the thermophylic strains of Lactobacillus.
(*) Corresponding author.
106
SELECTED CHARACTERISTICS OF SEVERAL...
Lactobacillus species are among the most widely used lactic acid bacteria, participating in the
production of food derivatives of diverse origin (meats, vegetables, milk, etc.). Due to the diversity
in both strains and processes, there is an increasing trend towards the use of carefully selected
strains with the most suitable characteristics for the different fermentations (3). One of the species
belonging to this genus, Lactobacillus plantarum, is a rod-shaped bacterium frequently found in
many cheese types. In Cabrales cheese, L. plantarum may account for up to 78 % of the total microflora at certain stages, suggesting that it may have a significant role on cheese maturation (25, 26).
In the present study, we have performed the characterization of several relevant properties of nine
strains of L. plantarum isolated from Cabrales cheese produced from raw milk, without the addition
of starter bacteria. The results obtained may help to understand the role of these bacteria in food
fermentations, where they often appear in significant numbers.
Materials and methods
Organisms
Eigth isolates of L. plantarum were obtained from Cabrales cheese at an early stage of maturation (5 d). The identification of strains was made according to Nunez and Medina (26) and Sharpe
(31). L. plantarum INIA 169, also isolated from Cabrales cheese, was kindly provided by M. Nunez.
Escherichia coli JM 83 (34) was donated by J. E. Suárez. Long term conservation of the strains without appreciable loss of properties was achieved by freezing concentrated suspensions of cells at
-20° C in MRS medium (Oxoid) supplemented with 50 % glycerol.
Media and culture conditions
Lactobacilli were incubated as static cultures at 30° C in MRS medium, unless otherwise indicated. Lactose indicator agar (22) and milk agar medium (11) were used as test media for detection
of lactose fermentation. E. coli cultures were prepared in LB medium .
Enzyme assays
For the determination of lactose hydrolyzing enzymes, cells were grown in lactose broth (21),
harvested by centrifugation, washed twice with 0.1 M phosphate buffer pH 7.0 and suspended in the
same buffer. Crude extracts were prepared by mechanical disruption with glass beads (0.1 mm diameter) in a Vibrogen homogenizer, at 0° C for 2 min. After centrifugation (12,000 xg, 15 min, 4° C),
the resulting supernatants were used as the enzyme source. 6-galactosidase (6-Gal) activity was estimated according to Citti et al. (9) with the chromogenic substrate o-nitrophenyl-6-D-galactopyranoside. 6-phosphogactosidase (B-PGal) was assayed with o-nitrophenyl-6-D-galactopyranoside-6phosphate, following the procedure of McKay et al (22). A unit of activity is defined as the
amount of enzyme needed for the production of 1 |imol of o-nitrophenol per minute. The activities
were normalized with the protein content of the crude extracts, measured by the method of Lowry
etal{lS).
Proteolytic activity was determined in whole cells previously grown and inoculated in skim
milk as described by Church et al (7). After 24 h of incubation, the amino groups released during
proteolysis were measured spectrophotometrically after derivatization with q-phthalaldehyde (8).
Results were expressed as the increase in absorbance at 340 nm with respect to an uninoculated
control.
B. MAYO, C. HARDISSON AND A. R BRAÑA
107
Plasmid profiles
Cell lysis and extraction of plasmids were done as described by Anderson and McKay (1). Purification by CsCl-ethidium bromide gradients was also performed in some cases. For the determination of plasmid profiles in E. coli transformants, the procedure of Holmes and Quigley (15) was
used. Electrophoresis were carried out in 0.7 % agarose gels.
Plasmid curing
L. plantarum LL2, that showed a high content of extrachromosomal DNA, was submitted to
several treatments often employed to induce plasmid loss. For temperature-induced curing, cells
were repeatedly subcultured at 45° C. Treatments with acriflavine, acridine orange and ethidium
bromide were performed as described by Caro et al (5). In every case, the cells were plated on lactose indicator agar, Elliker medium (12) supplemented with 20 ^g/ml 5-bromo-4-chloro-3-indolyl-6D-galactopyranoside (X-gal), and citrate indicator medium (16).
Cloning techniques
Extraction of plasmids from E. coli was done by the method of Birnboim and Doly (2). Plasmids were obtained from agarose gels by electroelution (20). Enzymes employed in DNA manipulations were from Boehringer Mannheim. DNA restrictions and ligations were done according to
manufacturer instructions. Transformation of E. coli JM83 was done as described by Hanahan (14).
Transformants were selected in LB plates with ampicillin (100 |Xg/ml) and X-gal (20 |ig/ml) (34).
Antimicrobial agent susceptibility
Disk tests and MIC determinations were done following standard procedures (17), with cells
previously grown in MRS medium . The compounds tested and disk contents are listed in Table 3.
Results
All L. plantarum strains studied were lactose fermenters when tested on lactose indicator agar
or milk agar medium. High B-Gal activities were detected in crude extracts, together with moderate levels of 6-PGal (Table 1). In an attempt to localize the genes encoding these activities, the
chromosomal and plasmid DNA of these strains was submitted to hybridization with a genetic
probe containing the B-PGal gene from Lactococcus lactis subsp. lactis MG 1299 (Mayo, B. et al
1989, J. Dairy Res., in press), and with plasmid pUC19 that contains the 6-Gal gene from E. coli.
No positive hybridization was observed in any case (data not shown).
No significant proteolytic activity was detected in any of the strains (Table 1). Values obtained
were lower than 5 % of the activities determined in proteolytic strains of L. lactis subsp. lactis using
the same method (Mayo, B. et al 1989, J. Dairy Res., in press). Furthermore, washed suspensions
of L. plantarum were unable to clot milk, except for strains LL441 and LL442 that produced coagulation after 5 d of incubation (Table 1). The proteolytic activity and milk clotting ability did not increase when the milk was supplemented with glucose, but the addition of a readily utilizable source
of amino acids (peptone plus yeast extract) allowed coagulation of milk in two days (Table 2).
108
SELECTED CHARACTERISTICS OF SEVERAL.
TABLE 1
LACTOSE FERMENTATION, PROTEOLYTIC ACTIVITY AND MILK CLOTTING ABILITY
OF L. PLANTARUM STRAINS
Specific activity
(mU/mg protein)
Strain
INIA 169
LLl
LL2
LL3
LL7
LL31
LL232
LL441
LL442
6-Gal
B-PGal
703.1
447.5
541.7
480.6
885.7
86.9
744.2
1,292.0
1,111.0
53.2
18.0
33.7
40.2
44.7
24.3
40.0
53.0
47.7
Proteolytic
Milk clotting
time (d)
0
0
0.006
0
0.008
0.003
0.006
0.005
0
>10
>10
>10
>10
>10
>10
>10
5
5
* Expressed as absorbance increase at 340 nm, as described in materials and methods.
Results on antimicrobial agent susceptibility are shown in Table 3. Except for lincomycin, the
strains showed a homogeneous behaviour with the compounds tested. Disk susceptibility assays indicated low sensitivity to several agents, but the levels of resistance, as measured in MIC tests, were
lower than those described for strains bearing specific resistance factors (4).
The presence of plasmid bands, ranging in size from 0.8 to 110 Mdal, was detected in all strains
(Table 4). Despite the similarity in the results obtained with most of the properties studied, plasmid
profiles differed between strains, except in the case of LL441 and LL442, which may represent a repeated isolation of a single strain present in the cheese.
When cultures of strain LL2 were plated after different plasmid curing treatments, and incubated in aerobiosis, colonies significantly smaller (0.1-0.5 mm in diameter instead of 1-2.5 mm of normal colonies) appeared at frequencies from 1 % to 2.5 %. These colonies did not cause a color
change in the indicator media used for screening. However, under anaerobic conditions (hydrogencarbon dioxide atmosphere), all colonies grew to a similar size and appeared to utilize lactose and
citrate in the respective indicator media. Twelve of the small colonies observed in aerobiosis were
isolated and their plasmid contents analyzed after cell lysis and purification in CsCl gradients. Plasmid losses were detected in eleven of these derivative strains (Table 5). When they were compared
TABLE 2
EFFECT OF SUPPLEMENTS ON PROTEOLYTIC ACTIVITY AND MILK CLOTTING ABILITY
OF L. PLANTARUM STRAINS
Supplement
Strain
LL2
LL7
LL442
0.5 % Glucose
0.125 % peptone + 0.125 % Yeast extract
Proteolysis *
Milk clotting (d)
Milk clotting (d)
0
0.002
0.004
>10
>10
5
2
2
2
Expressed as absorbance increase at 340 nm, as described in materials and methods.
B. MAYO, C. HARDISSON AND A. R BRAÑA
109
TABLE 3
ANTIMICROBIAL AGENT SUSCEPTIBILITY OF L. PLANTARUM STRAINS
Disk tests *
Antimicrobial agent **
Nalidixic acid (30)
Sulfadiacine (300)
Streptomycin (10)
AmpiciUin (10)
Kanamycin (30)
Gentamycin (10)
Tetracycline (30)
Chloramphenicol (30)
Vancomycin (30)
Lincomycin (2)
Cephalotin (30)
Fosfomycin (50)
Penicillin G (10)
Rifampicin
MIC (^ig/ml)
Resistant
Sensitive
Mode
Highest
9
9
9
0
9
9
0
0
0
5
9
9
9
ND
0
0
0
9
0
0
9
9
9
4
0
0
0
ND
32
100
64
2
32
32
ND
4
ND
ND
32
64
32
4
64
500
64
2
32
32
ND
4
ND
ND
32
64
32
4
* Number of resistant or sensitive strains are given. Interpretation of inhibition zones was done according to
Lennette et al. (17).
** The numbers in brackets indicate the disk content in (Xg.
ND: Not determined.
TABLE 4
PLASMID PROFILES OF L. PLANTARUM
STRAINS
Strain
INIA 169
LLl
LL2
LL3
LL7
LL31
LL232
LL441
LL442
Plasmids (Mdal)
30; 16.6; 12; 8; 6.6; 2; 1.4
34; 12; 8.6; 4.8; 2; 1.4; 0.9
110; 70; 60; 50; 44; 30; 18; 6; 1.2
30; 21; 18; 12; 4.6; 1.4; 0.9
50; 5.8; 4.6
50; 30; 18; 1.2; 0.8
50; 8; 4.8
30; 5.5; 3.7
30; 5.5; 3.7
with their parent strain (LL2) using the API 50 test, no change in the physiological properties examined was observed. Furthermore, the pattern of antimicrobial agent susceptibility, the 6-Gal and
6-PGal levels, and the production of diacetyl, remained unaltered in the cured strains.
Due to the high copy number and the stability observed in curing experiments, the 1.2 Mdal
plasmid from strain LL2, designated pLL21, was selected for cloning into the E. coli plasmid
pUC19. Both plasmids had a single Eco RI site and, after digestion with this enzyme, they were mixed, ligated «in vitro», and the ligation mixture used to transform competent cells of E. coli JM 83.
After selection of the transformants, one of them contained a new plasmid with the expected size
110
SELECTED CHARACTERISTICS OF SEVERAL...
TABLE 5
DERIVATIVE STRAINS OF L. PLANTARUM LL2 OBTAINED WITH CURING AGENTS
Strain
Screening medium *
Cured plasmids (Mdal)
New plasmids (Mdal)
A
A
B
C
C
C
44
50; 44
44
60; 50
110
60
—
—
—
26
—
—
:, LL23, LL24
\ LL28, LL29, LL210
1
2.
* The screening media used were: A, lactose indicator agar; B, EUiker medium + X-gal; C, citrate indicator
medium.
for a hybrid pUC19-pLL21 plasmid. This clone was purified and its plasmid D N A utilized for a new
transformation of E. coli JM 83. The transformants contained the new plasmid t h a t , after extraction and purification in CsCl gradients, was physically mapped with several restriction enzymes
(Fig. 1). The 1.2 Mdal fragment inserted in pUC19 had restriction sites coinciding with pLL21,
which confirmed the identity of the recombinant plasmid.
Discussion
All the L. plantarum strains studied here were efficient lactose fermenters and they appeared
to use both the B-Gal and 6-PGal pathways, a result previously found by Premi et al (28) with one
strain of this species. Unfortunately, the degree of homology between the genes coding for the galactosidase activities and the probes used for hybridization, was not enough to allow their localization. The probe used for the 6-PGal gene had been previously found to hybridize with D N A from
different Lactococcus lactis strains that present this activity (Mayo, B. et al 1989, J. Dairy Res., in
PvuII
EcoRI
Clal
Hind III
"Âva I
—
PvuII
Fig. 1. Restriction endonuclease map of the hybrid plasmid comprising pUC19 (thin line) and pLL21 (thick une).
B. MAYO, C. HARDISSON AND A. R BRAÑA
111
press). Therefore, a significant interspecific genetic diversity among lactic acid bacteria appears to
exist, at least concerning this gene.
Since L. plantarum strains can catabolize lactose and acidify culture media with this sugar as
the carbon source, their inability to coagulate milk seems to be due to some nutritional requirement
not present in milk but provided by the supplement of peptone plus yeast extract. The existence of
such a requirement might also explain the lack of significant proteolytic activity after inoculation in
skim milk, simply due to the inability of the cells to grow in this medium. Alternatively, there may
be a true deficiency in their proteolytic system, which would be compatible with all these results.
Whatever the explanation is, L. plantarum is present at high numbers in Cabrales cheese (25, 26).
Therefore, it seems that the metabolic activities of other members of the cheese microflora would
meet L. plantarum requirements (amino acids or other nutrients), and this bacterium could make a
contribution to cheese maturation through their lactose fermenting ability and the production of
compounds involved in flavour and aroma.
Resistance to high levels of antimicrobial agents does not seem to be a common trait in lactic
acid bacteria (27, 29, 30). However, the moderate levels of resistance found in L. plantarum against
antibiotics such as cephalotin and penicillin G, may be useful for its selective isolation from mixed
populations of lactic acid bacteria. Growth of both Lactococcus and Leuconostoc species was inhibited by concentrations of these antibiotics much lower than those needed to inhibit L. plantarum
(Mayo, B. 1988, Ph. D. thesis. Universidad de Oviedo).
The presence of small cryptic plasmids in strains of L. plantarum has been reported by other
authors (24, 32, 33). This was also observed in our isolates, but the use of the lysis method of Anderson and McKay (1) allowed the detection of plasmids with a wide range of molecular weights. In
other lactic acid bacteria, mainly lactococci, many of their relevant properties are encoded by extrachromosomal DNA (23). However, our attempts to relate the presence of plasmids in L. plantarum with a particular characteristic have not been successful. The maintenance of such an amount
of plasmid DNA must have a high metabolic cost, and therefore it is reasonable to suposse that these plasmids contain some advantageous information for growth and survival under natural conditions, but that may not be obvious in laboratory conditions. In any case, the poor growth of our cured strains under aerobic conditions suggests that some plasmids may have a direct or indirect role
on oxygen tolerance.
The application of recombinant DNA technology to the majority of Lactobacillus species lags
well behind the situation for lactococci, although recent efforts have been made to close this gap (6,
10,13, 19, 33). In the case of L. plantarum, no efficient cloning vectors have been developed to the
present date. The recombinant plasmid obtained in this work, providing that a suitable marker is introduced to allow selection in L. plantarum, constitutes a starting point for the construction of a
useful shuttle vector between E. coli and L. plantarum.
Acknowledgments
This work was supported by a grant from the Fondo para la Investigación Científica y Técnica
(FICYT). B. Mayo was the recipient of a fellowship from the Principado de Asturias/CSIC.
References
1. Anderson, D. G. and McKay, L. L. (1983). Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl. Environ. Microbiol. 46,549-552.
2. Birnboim, H. C. and Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant DNA. Nuc. Acids
Res. 7,15134523.
112
SELECTED CHARACTERISTICS OF SEVERAL...
3. Botazzi, V. (1988). An introduction to rod-shaped lactic acid bacteria. Biochimie 70,303-315.
4. earlier, C. and Courvalin, P. (1982). Resistance of streptococci to aminoglycoside-aminocyclitol antibiotics. In: D. Schlessinger (éd.). Microbiology, 1982, pp. 162-166. American Society for Microbiology, Washington DC.
5. Caro, L., Charchwars, G. and Candler, M. (1984). Study of plasmid replication «in vivo». In: P. M. Bennett and J. Grinsted (eds.). Methods in Microbiology, vol. 17, pp. 97-122. Academic Press, New York.
6. Chassy, B. M. (1987). Prospects for the genetic manipulation of lactobacilli. FEMS Microbiol. Rev. 46,297-312.
7. Church, F. C , Swaisgood, H. E., Porter, D. H. and Catignani, G. L. (1983). Spectrophotometric assay using o-phthaldialdehyde for determination of proteolysis of milk and isolated milk proteins. J. Dairy Sci. 66,1219-1227.
8. Church, F. C , Porter, D. H., Catignani, G. L. and Swaisgood, H. E. (1985). An o-phthaldialdehyde spectrophotometric
assay for proteinases. Anal. Biochem. 146,343-348.
9. Citti, J. E., Sandine, W. E. and EUiker, P. R. (1965). Comparison of slow and fast acid producing Streptococcus lactis.
J. Dairy Sci. 48,14-18..
10. Cosby, W. M., Casas, J. A. and Dobrogosz, W. J. (1988). Formation, regeneration and transfection of Lactobacillus plantarum protoplasts. Appl. Environ. Microbiol. 54,2599-2602.
11. Efstathiou, J. D. and McKay, L. L. (1976). Plasmids in Streptococcus lactis: evidence that lactose metabolism and proteinase activity are plasmid linked. Appl. Environ. Microbiol. 32,38-44.
12. EUiker, P. R., Anderson, A. W. and Hannesson, G. (1956). An agar culture medium for lactic acid streptococci and lactobacilli. J. Dairy Sci. 39,1611-1612.
13. Fitzgerald, G. F. and Gasson, M. J. (1988). «In vivo» gene transfer systems and transposons. Biochimie 70,489-502.
14. Hanahan, D. (1985). Techniques for transformation of E. coli. In: M. Glover (éd.). DNA cloning, vol. I, pp. 109-135. IRC
Press, Oxford and Washington DC.
15. Holmes, D. and Quigley, M. (1981). A rapid boihng method for the preparation of bacterial plasmids. Anal. Biochem.
144,193-197.
16. Kempler, G. M. and McKay, L. L. (1980). Improved medium for detection of citrate fermenting Streptococcus lactis
subsp. diacetylactis. Appl. Environ. Microbiol. 39,926-927.
17. Lennette, E. H., Balows, A., Hausler, W J. Jr. and Shadomy, H. J. (1985). Manual of Clinical Microbiology, 4th ed. American Society for Microbiology, Washington DC.
18. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193,265-275.
19. Luchanski, J. B., Muriana, P. M. and Klaenhammer, T. R. (1988). Application of electroporation for transfer of plasmid
DNA to Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Bacillus, Enterococcus and Propionibacterium.
Mol. Microbiol. 2,637-646.
20. Maniatis, T., Frisch, E. F and Sambrook, J. (1982). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
21. McFeters, G. A., Sandine, W E. and EUiker, P. R. (1967). Purification and properties of Streptococcus lactis B-galactosidase. J. Bacteriol. 93,914-919.
22. McKay, L. L., MUler, A., Sandine, W. E. and EUiker, P. R. (1970). Mechanisms of lactose utilization by lactic streptococci:
enzymatic and genetic analysis. J. Bacteriol. 102,804-809.
23. McKay, L. L. (1983). Functional properties of plasmids in lactic streptococci. Antoine van Leeuwenhoek 49,259-274.
24. Nes, I. F. (1983). Plasmid profiles of ten strains of Lactobacillus plantarum. FEMS Microbiol. Lett. 21,359-361.
25. Nunez, M. (1976). Microflora of Cabrales cheese: changes during maturation. J. Dairy Res. 45,501-508.
26. Nunez, M. et Medina, M. (1979). La flore lactique du fromage bleu de Cabrales. Le Lait 59,497-513.
27. Orberg, P. K. and Sandine, W. E. (1985). Survey of antimicrobial resistance in lactic streptococci. Appl. Environ. Microbiol. 49,538-542.
28. Premi, L., Sandine, W. E. and EUiker, P. R. (1972). Lactose-hydrolyzing enzymes of Lactobacillus species. Appl. Microbiol. 24,51-57.
29. Reimbold, G. W and Reddy, M. S. (1974). Sensitivity or resistance of dairy starter and associated microorganisms to selected antibiotics. J. Milk Food Technol. 37,517-521.
30. Sinha, R. P. (1986). Development of high-level resistance affected by a plasmid in lactic streptococci. Appl. Environ. Microbiol. 52,255-261.
31. Sharpe, M. E. (1981). The genus Lactobacillus. In: M. R Starr, H. Stolp, H. G. Trupper, A. Bariows and H. G. Schlegel
(eds.). The Prokariotes, pp. 1653-1679. Springer-Verlag, Berlin and Heidelberg.
32. Von Husby, K. O. and Nes, I. F. (1986). Changes in the plasmid profile of Lactobacillus plantarum obtained from commercial meat starter cultures. J. Appl. Bacteriol. 60,413-417.
33. West, C. A. and Warner, P. J. (1985). Plasmid profiles and transfer of plasmid-encoded antibiotic resistance in Lactobacillus plantarum. Appl. Environ. Microbiol. 50,1319-1321.
34. Yanisch-Perron, C , Vieira, J. and Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide
sequences of the M13mpl8 and pUC19 vectors. Gene 33,103-119.
MICROBIOLOGÍA SEM
5 (1989), 113-119
The effect of Trichoderma viride C-1 UV
mutagenization on celiulases activity
Danuta Witkowska^ Malgorzata Bien^ and Jerzy Sobieszczanski*
^ Department of Biotechnology and Food Microbiology. Academy of Agriculture. 50-375 Wroclaw, Norwida 25, Poland.
^ Institute of Microbiology, University of Wroclaw, 51-148 Wroclaw, Pnybyszewskiego 63/77, Poland
(Received June 17,1988/September 15,1989)
Summary
Trichoderma viride C-1 strain was irradiated with UV until the survival level of 0.03 % was obtained. Sixteen mutants were isolated on the basis of the visible clearance zone around the colonies
on the media with cellulose and glycerol. Then they were cultivated on a rotary shaker in the liquid
Saunders medium supplemented with microcrystalline cellulose and comminuted sugar beet pulp.
Exo-l,4-p-glucanase, endo-l,4-p-glucanase and p-glucosidase were assayed in the supematants of
postcultural liquids at different time intervals of culture. The same mutants were characterized by
higher biosynthesis level of exoglucanase (1.2-5.0 times) endoglucanase (1.2-2.5 times) and p-glucosidase (1.5-1.7 times) when compared with the wild type strain.
Keywords: Mutants, Trichoderma, celiulases.
Resumen
La cepa Cl de Trichoderma viride fue irradiada con luz ultravioleta hasta que se obtuvo el nivel de supervivencia del 0,03.%. Se aislaron 16 mutantes que formaron un halo translúcido alrededor de las colonias en medios con celulosa y glicerol. Los mutantes fueron cultivados en un agitador
orbital en medio líquido Saunders enriquecido con celulosa microcristalina y pulpa de remolacha
azucarera pulverizada. Las actividades exo-l,4-P-glucanasa, endo-l,4-p-glucanasa y p-glucosidasa se
determinaron en los sobrenadantes de los cultivos a diferentes intervalos de tiempo. Los mutantes
se caracterizaron por un nivel más alto de biosíntesis de exoglucanasa (1,2-5,0 veces), endoglucanasa (1,2-2,5 veces) y p-glucosidasa (1,5-1,7 veces) cuando se compararon con la cepa salvaje.
Introduction
The use of enzyme preparations of high activity is one of methods for reducing the costs of industrial hydrolisis of cellulose substrates. For this reason, in recent years much attention .has been
drawn to mutagenization of microorganisms in order to obtain the strains of high performance (3,4,
6,10,11,13,14,18). Mutans with enhanced exo-1.4-p-glucanase activity contributing to degradation
(*) Corresponding author.
114
THE EFFECT OF TRICHODERMA VIRIDE...
of native cellulose are of particular value. In the present study, a wild strain Trichoderma viride C-1
was mutagenized using UV irradiation and mutants with enhanced cellulase activity were selected.
Materials and methods
Microorganism
The wild strain Trichoderma viride C-1 from the Department of Biotechnology and Food Microbiology at the Agricultural Academy in Wroclaw was used for the UV induction of mutants.
Media
Five different media were prepared. The solid Sabouraud (5) medium was used for cultivation
and preservation. The cells were UV irradiated on a mineral agar medium with a trace element solution II according to Theodorou (20) supplemented with 33.3 |ig/ml of rose bengal. The above medium but supplemented with 1 % cellulose type He veten 10 (DDR) instead of glucose was used for
screening and selection. For the same purpose we used the above medium supplemented with 5 %
glycerol and 1 % microcrystalline cellulose (Merck) (11).
The submerged cultures of the examined strains were carried out in the Saunders medium (15)
containing 2 ^% microcrystaUine cellulose (Merck), 2 % beet pulp 0.5 % yeast extract (Difco), 0.5 %
wheat germs and 0.1 % glucose.
Induction and selection ofmutans
Mutants were inducted by UV irradiation. Water suspension (with 0.1 % Tween 80) of 4.9 x 10"*
conidia free of myceha per plate (100 plates) obtained from 7 day cultures on Sabouraud plates were irradiated with UV Hanau Quarzlitat lamp (7.1 erg/mm^/sec.) from a distance of 20 cm for 90 seconds. The survival level was 0.03 %. The 1,400 clones which survived UV irradiation were isolated
on a plate on Theodorou medium (20) supplemented with glucose. The clones were incubated at
28° C for 3 days and then replicated on the selecting media as above. The replica plates were incubated at 28° C 3 days and then at 50° C for 24 h in order to intensify the clearing zone around the
colonies on the media with cellulose and glycerol.
Further screening based on excretion of extracellular hydrolases, was done by growing the selected mutants and the wild type strain at 28-30° C for 144 h in the Saunders liquid medium on a rotary shaker. The experiments in triplicate were carried out in 500 ml Erlenmeyer flasks with 100 ml
Saunders medium inoculated with 5 x 10^ conidia. The culture liquid was maintained at pH=5.0 by
means of sterile IN HCI or IN NaOH. After 2 days of incubation, samples were taken from the
flasks each 24 h in order to assay the enzymatic activity and protein.
Enzyme assay
Exo-1.4-p-gIucanase activity was determined at 50° C, pH=4.8 using strips ( 1 x 6 cm) of Whatman No. 1 (8). Endo-1.4-P-glucanase activity was determined at 50° C, pH=4.8 using CMCNa as a
substrate in 0.05M citrate buffer (8). One unit enzyme activity in these assays is defined as the
amount of enzyme which produced one ^imol of reducing sugar per minute per millilitre of culture
D. WITKOWSKA, M. BIEN AND J. SOBIESZCZANSKI
115
fihrate. Reducing sugar was estimated colorimetrically using dinitrosahcyhc acid reagent (9). p-glucosidase activity was determined at 37° C, pH=3.2 using cellobiose (Merck) as a substrate (16). One
unit of p-glucosidase activity is defined as the amount of enzyme which catalyses the formation of
one ^mol of glucose per minute, per miUihtre of culture filtrate. The released glucose was estimated
by the glucose-oxidase-peroxidase-reaction (Fermognost, Veb Laborchemie Apolda, DDR).
Proteolytic activity was determined according to Anson (2). Soluble proteins were determined
in the culture according to Lowry (7).
Cell proteins were determined by subtraction of extracellular proteins (Lowry) from quantity
of total culture proteins [Kjeldahl, N(protein) x 6.25]. The content of cell proteins was used as a
rough index of mycelium growth.
Results and discussion
After the mutagenization with UV irradiation of a wild T. viride C-1 strain, 49 clones were isolated from the 1,400 survivals. Next, 16 clones were isolated from the above 49 clones, and these exhibited different zones of visible clearance around them on cellulose solid medium.
In the submerged culture, the examined mutants and the wild strain showed different P-glucanase activity (Fig. 1).
The greatest changes in the activity of the examined enzymes were observed in exo-glucanases,
since 1.2-5 fold increase in their specific activity was obtained (0.2-0.79 U/mg) as compared with the
wild strain (0.16 U/mg). Oh the other hand, the mutants M4-5 and M7-1 proved to be the best pro-|
ducers of exo-glucanases (0.79 and 0.68 U/mg) (Fig. 2).
In the case of endo-glucanases, the ability to their biosynthesis was twice higher in 50 % of the
UV mutants, whereas M4-5, M4-6 and M7-1 mutants exhibited the highest activity (1.7,1.78 and 1.8
M4-1
MA-4
MA-51
M4-6
M4-7
M5-1
M5-3
M5-4I
M5-5
M5-6I
M5-7
M7-1
M7-3
M7-4
M7-6
M7-7
C-1
—I
0.2
1
1
1—'
1
1
1
\
1 —
0.6
1,0
1.4
1.8
/Specific endoglucanase activity [ U/mg]
Fig. 1. Specific endo-glucanase activity of wild strain. T. viride C-1 and its UV mutans after 96 hours cultivation.
116
THE EFFECT OF TRICHODERMA VIRIDE...
MA-1
M4-4!
M4-5|
MA-7¡
M5-1J
M5-3
M5-A!
M5-5
M5-6
M5-7
M7-1!
M7-3|
M7-4J
M7-6
M7-7I
C-1
—r-
0,1
0.3
0,5
0,7
Specific exoglucanase activity
0,9
[ U/mg]
Fig. 2. Specific exo-glucanase activity of wild strain. T. viride C-1 and UV mutans after 96 hours cultivation.
U/mg, respectively) as compared with the wild strain (Fig. 1). The increased p-glucosidase biosynthesis was obtained only in the case of two mutants M4-1 and M7-1. Specific activities of |3-glucosidases (0.11 U/mg and 0.095 U/mg) of these mutants were 1.5-1.8 fold higher than those of the wild
strain. The other mutans showed their enzyme activity decreased by 6-90 % (Fig. 3).
MA-1
MA-41
MA-5
MA-el
MA-7
M5-1
M5-3
M5-A
M5-5|
M5-6|
MS-7
M7-1
M7-3
M7-A
M7-6|
M7-7I
C-1
0.02
O.OA
0.06
0.08
0.10
Specific '&-glucosidase activity
[ U/mg 1
Fig. 3. Specific p-glucosidase activity of wild strain. T. viride C-1 and its UV mutans after 96 hours cultivation.
D. WITKOWSKA, M. BIEN AND J. SOBIESZCZANSKI
117
TABLE 1
EXTRACELLULAR HYDROLASE ACTIVITY OF MILD STRAIN T VIRIDEC-1 AND ITS UV
MUTANT M-7-1 IN SUBMERGED CULTURE AFTER 96 HOURS.
Exo-glucanase
Strains
U/ml
C-1
M7-1
0.38 ±0.03
2.71 ± 0.23
U/mg
0.16
0.68
Endo-glucanase
U/ml
1.74 ±0.16
7.2 ±0.46
p-glucosidase
Proteinase
Protein
U/mg
U/ml
U/mg
U/ml
U/mg
mg/ml
0.72
1.8
0.155 ±0.014
0.38 ± 0.02
0.064
0.095
1.4 ±0.09
0.72 ±0.03
0.58
0.18
2.4 ±0.11
4.0 ±0.25
The results obtained in the study indicate that the UV mutants T. viride C-1 especially mutant
M7-1 were similar in some cellulolytic activities of other kinds of fungi obtained by other authors
(Table 1).
Zaldivar et al (21) obtained a B^ mutant pseudokoningi by NG mutagenization. It exhibited
three times higher exoglucanase (from 0.14 to 0.44 U/ml) and endo-glucanase (from 5.3 U/ml to
15.9 U/ml) but 1.5 times higher p-glucosidase activity (from 0.13 U/ml to 0.2 U/ml) than the wilds
type strain.
The mutant T. reesei after UV and NG mutagenization, obtained by Bland and Montenecourt
(3) exhibited a 5-fold increase in exo-l,4-|3-glucanase (4.65 U/ml), 2-fold increase in endo-l,4-p-glucanase (15 U/ml) and 4-fold increase in P-glucosidase activity (1.35 U/ml) in comparison with T. reesei QM 9414. Ostrikowa and Konowalow (12,13) obtained an active T viride 44 mutant as a result
of three-stage mutagenesis carried out in the presence of nitroso-dimethylurea, ethylenimine and
thermal shock at 100° C for 1 min. The mutant thus obtained exhibited four times as high exo-glucanase activity (22.2 mg/ml/h) as the wild strain (5.7 mg/ml/h).
Targonski (19) examined an UV mutant of Fusarium sp. FS-27 and found 1.7 fold increase in
endo-glucanase activity (from 12.4 |xmol/ml to 21.5 |xmol/ml, vise method) and 1.6 fold increase in
exo-glucanase (from 0.73 to 1.2 U/ml) and a decrease in p-glucosidase activity (from 0,25 ^imol/ml
to 0.12 |imol/ml) in comparison with the wild strain. The same mutant but in semicontinous culture
showed 2.4 fold increase in endo-glucanase activity (1).
Two out of the four UV mutants of T viride QM 9414 obtained by Farkas et al. (4), M6 and
MHC 22, showed 1.2-1.4 higher exo-glucanase but MHC15 and MHC 22 mutants showed 2.3-3.2
higher p-glucosidase activity than the parent strain.
In the present study, we also draw our attention to the kinetics of cellulase synthesis of UV mutant M7-1 and of the wild type T. viride C-1 strain. Figs. 4 and 5 show that maximal production occurs after 96 h of cultivation, i. e., at the end of the stationary phase of growth. In the next hours of
cultivation, cellulase activity gradually decreased and this coincided with the lowering curve of
growth.
The decreased cellulase activity in post cultural liquid (Fig. 4 and 5) at the final phase of
growth is difficult to explain. This decrease is affected by several factors, i. e. increasing pH, aeration of the culture or presence of proteolytic enzymes in the culture.
Farkas etal (4) explained that decreased cellulolytic activity at the end of cultivation of T. viride in submerged cultures could be attributed to inactivation of cellulases by the increased pH of the
cultures in the stationary phase of growth.
In our studies, the pH of the submerged cultures of T. viride C-1 and the mutans was controlled
each 24 h and maintained at pH=5.0 but in the course of time, until the next control after 24 h, the
pH increased to 6.0-7.0 which could also affect cellulase activity (Witkowska D., 1988 in preparation). The decreased cellulase activity at the final phase of growth of A. terreas has been explained
by Szczodrak (17), as an effect of proteolytic enzymes whose activity increased regularly during
their growth, as found in our experiments (Figs. 4 and 5).
118
THE EFFECT OF TRICHODERMA
VIRIDE..
Fig. 4. Dynamics of cellulolytic enzymes and
proteinase synthesis in submerged culture
of UV mutant M7-1 T viride:
4A: exo-glucanase (A), endo-glucanase (A)
P-glucosidase (o), proteinase (•),
4B: mycelial protein ( a ) , pH (x)
Fig. 5. Dynamics of cellulolytic enzymes and proteinase synthesis in submerged culture of wild strain T. viride C-1. Symbols as
in Fig. 4.
D. WITKOWSKA, M. BIEN AND J. SOBIESZCZANSKI
119
Out current studies on the mutans described here are directed to increase extracellular hydrolase, activities by several stages of mutation of the wild strain and to get detailed characteristics of
other hydrolases produced by the resultant mutants.
References
1. Achremowicz, B., Baraniak, A., Bujak, St. and Targonski, Zd. (1984). Biosynthesis of cellulolytic enzymes by strain Fusarium sp. 25/2 and its mutant Fusarium sp. 27 by the semicontinuous method on microtechnical scale, Acta Alimen, Polon. 10,357-363.
2. Anson, M. L. (1939). Gen Physiol. 22,79.
3. Bland, S., Montenecourt, B. S. and Eveleigh, D. E. (1977). Preparation of mutants of Trichoderma reesei with enhanced
cellulase production. Appl. Environm. Microbiol. 34,111-1%1.
4. Farkas, V, Labudova, L., Bauer, S. and Ferenczy, L. (1981). Preparation of mutants of Trichoderma viride with increased
production of cellulase. Folia Microbiol. 26,129-132.
5. Koch, M. (1981). Leitfaden der Medizinischen Mycologie, pp. 69, VEB Gustav Fischer Verlag (Jena).
6. Labudova, L, Farkas, V., Bauer, St., Kolarova, N. and Branyik, A. (1981). Characterization of cellulolytic Enzyme complex obtained from Mutans of Trichoderma reesei with enhanced cellulase production. Eur. J. Appl. Microb. Biotechnol.
12,16-21.
7. Lowry, J. G. H., Rosenbrough, N. I., Farr, A. Z. and Randall, R. I. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193,265.
8. Mandéis, M., Andreotti, R. and Roche, C. (1976). Measurement of saccharyfying cellulose. Biotechn. Bioeng. Symp. 6,
17-34.
9. Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chem. 31, 426428.
10. Mishra, S., Gopalkrishnank, S. and Ghosa, T. K. (1982). A constitutivel^ cellulase producing Mutant of Trichoderma reesei. Biotechn. Bioeng. 24,251-254.
11. Montenecourt, B. S. and Eveleigh, D. E. (1977). Semiquantitative plate assay for determination of cellulase production
by Trichoderma viride. Appl. Environm. Microbiol. 33,178-183.
12. Ostrikova, N. A. and Konowalow, S. A. (1980). Selekcja Trichoderma viride producenta celljulazy. Mikrobiologija, 49,
540-545.
13. Ostrikova, N. A. and Konowalow, S. A. (1980). Osobiennosti mutantnogo stamma Trichoderma viride 44 producenta
celljulazy. Prik. Biochm. Mikrobiol. 16,56-59.
14. Sadana, J. C , Shewale, J. G. and Deshpande, M. V. (1979). Enhanced cellulase production of Mutant Sclerotium rolfsi.
Appl. Environm. Microb. 38,730-733.
15. Saunders, P. R., Siu, R. G. K. and Genest, R. N. (1948). A cellulolytic enzyme preparation from Myrothecium verrucaria.
J. Biol. Chem. 174,697-703.
16. Schulz, G. and Hirte, W. F (1989). Special submers screening of cellulolytic fungi and selection of Pénicillium wild strain
for cellulase production with high substrate concentration. Zentralbl. Microbiol. 144,81-96.
17. Szczodrak, J., Rogalski, J. and Ilczuk, Zdz. (1984). Cellulolytic Activity of Moulds. IV. Evaluation of the utility of cellulosis wastes for biosynthesis of cellulases and xylanase by Aspergillus terreus F-413. Acta Microbiol. Polon. 33,217-225.
18. Tangu, S. K., Blanch, H. W. and Wilke, C. R. (1981). Enhanced production of cellulase, hemicellulase and p-glucosidase
by Trichoderma reesei. Biotechn. Bioeng. 23,1837-1849.
19. Targonski, Zd. (1983). Enhanced cellulolytic enzyme biosynthesis of Fusarium sp. strain by mutation and optymization of
culture conditions. Acta Microb. Polon. 32,153-159.
20. Theodoreu, M. K., Bazin, M. J. and Trinei, A. P. J. (1983). Growth of Trichoderma reesei on glucose mineral salts media
containing ammonium sulphate or urea as the source of nitrogen. Microbios 36,157-160.
21. Zaldivar, M., Steiner, J., Musalem, M. and Contreras, I. (1987). Isolation of a mutant of Trichoderma pseudokoningii with
enhanced cellulase production. Microbiología, SEM 3,33-44.
M I C R O B I O L O G Í A SEM
5 (1989), 121-125
NOTES
Saprophytic fungi on hair and feathers from apparently
healthy animals
M.^ Lourdes Abarca*, M.^ Rosa Bragulat, M.^ Teresa Bruguera and Francisco Javier Cabanes
Departamento de Patología y Producciones Animales (Microbiología). Facultad de Veterinaria. Universidad Autónoma de
Barcelona. Bellaterra, Barcelona. Spain
(Received February 14/March 15,1989)
Summary
The occurrence of saprophytic fungi on hair and feathers samples taken from apparently healthy domestic animals (cows, pigs, rabbits, and chickens) has been studied. A total of 221 strains
classified in 24 genera were isolated. The most frequent genera were Aspergillus, Pénicillium, Mucor, Absidia and Alternaría, and some statistically significant differences of contamination were detected among the animal species.
Key words: Mycoflora, hair, feathers, healthy animals.
Resumen
Se ha realizado un estudio de los hongos presentes en muestras de pelos y plumas de animales
aparentemente sanos (vacas, cerdos, conejos y gallinas). Se han aislado 221 cepas pertenecientes a
24 géneros. Los géneros aislados más frecuentemente fueron Aspergillus, Pénicillium, Mucor, Absidia y Alternaría, y se obsevaron diferencias significativas al comparar la frecuencia de aparición de
los distintos géneros y especies fúngicas en las especies animales estudiadas.
The hair and feathers of animals represent a suitable substrate for the colonization of keratinophilic fungi, some of which are pathogens, opportunistic pathogens or allergens. Studies focused
on the knowledge of mycoflora of this substrate have gradually revealed that the findings of fungi in
this kind of samples may reflect specific relationships between the fungi and their hosts.
It is not known whether most of the saprophytic fungi can produce infections in healthy animals. However, some of them become invasive in conditions of decreased resistance, thus being opportunistic in their pathogenicity. Cases of suspected fungal infections caused by normally saprophytic fungi have been described in animals during the last two decades (1). However, only a
few surveys about the fungal flora of asymptomatic skin of animals have come out (1,2,6, 8,11,14).
In this work we study the occurrence of saprophytic fungi on hair and feather samples taken from
apparently healthy domestic animals.
Seventy breeding places in Cataluña, Spain, have been sampled (20 of cows, 20 of pigs, 15 of
(*) Corresponding author.
122
SAPROPHYTIC FUNGI ON HAIR..
rabbits and 15 of chickens). It was taken hair or feather samples of one selected animal from each
breeding place. Samples were placed on the surface of Sabouraud's dextrose agar with chloramphenicol (DIFCO) and incubated at 28° C. Three plates per animal were used. Cycloheximide was not
used because its selective influence on the growth of contaminant moulds and yeasts. The taxonomic identification was performed according to the macro- and microscopic morphology of the suspected colonies. All the strains were cultivated on the appropriate culture medium to achieve typical growth and sporulation and identified according to the criteria estabUshed by the mycological
handbooks published for each genera. The yeast colonies were identified using the API 20 C AUX
system. The x^ test was used when possible to determine if there were significant differences of contamination among the animal species.
A total of 221 strains classified in 24 genera were isolated during the study. Figure 1 shows the
percentage occurrence of the 10 most frequent genera arranged in accord with the descending frequency of occurrence. All the dematiaceous strains which did not sporulate under laboratory conditions within 30 days, are grouped together as «dematiaceous sterile mycelia».
Fig. 1. Percentage occurrence of the 10 most frequent genera (including dematiaceous sterile mycelia) isolated from cow's hair
(1), pig's hair (2), rabbit's hair (3) and chicken's feather (4).
**: Statistically significant differences (x^test) p < 0.01.
Table 1 shows the various species of fungi isolated and the frequency of their occurrence
among the animal species studied. In both cases, the significant differences among the animal species (%^ test) are pointed out.
The most frequent genera were Aspergillus, Pénicillium, Mucor, Absidia and Alternaria. All
these genera are ubiquous in nature and nearly all the isolated genera have been reported in similar
studies made on asymptomatic animals. In a survey of the normal fungal flora of dogs (14), the authors reported that Cladosporium and Alternaria were the commonest genera isolated. Other studies
made in asymptomatic dogs (2, 8) showed the isolation of some dermatophyte species and strains of
genera Scopulariopsis, Pénicillium, Aspergillus and Alternaria among others. Marsella et al. (11)
studying the occurrence of fungi in animals of the Zoological Park of Rome reported among the ac-
M." L. ABARCA, M.' R. BRAGULAT, M.' T. BRUGUERA AND R J. CABANES
123
TABLE 1
FREQUENCY OF FUNGAL SPECIES OCCURRENCE AMONG THE ANIMALS EXAMINED
Fungal species
Absidia corymbifera
Acremonium strictum
Acremonium terrícola
Alternaría alternata **
Alternaría longípes
Alternaría tenuíssíma
Aphanocladíum album
Artríníum phaeospermum
Arthroderma sp.
Aspergillus amstelodamí
Aspergillus awamorí
Aspergillus candidas
Aspergillus chevalieri
Aspergillus flavus
Aspergillus fumigatus
Aspergillus nídulans
Aspergillus terreus
Aspergillus terrícola
Aspergillus tubíngensis *
Aspergillus versicolor
Candida famata
Candida lusitaníae
Circínella umbellata
Cladosporium dadosporioides
Cladosporium herbarum
Cladosporium macrocarpum
Epicoccum purpurascens
Fusarium oxysporum
Fusarium equiseti
Geotrichum candidum
Gliodadium catenulatum
Gliodadíum virens
Mucor circinelloides
Mucor hiemalis
Mucor plumbeus
Mucor racemosus
Paecilomyces variotii
Pénicillium carneo-lutescens
Pénicillium chrysogenum
Pénicillium frequentans
Pénicillium fellutanum
Pénicillium jensení
Pénicillium martensii
Pénicillium olivino-víride
Pénicillium palitans
Pénicillium variabile
Pénicillium víridicatum
Cows
n = 20
10
2
Pigs
n = 20
Rabbits
n = 15
Chickens
n = 15
124
SAPROPHYTIC FUNGI ON HAIR...
TABLE 1 (CONTINUED)
Fungal species
Phoma glomerata
Rhizopus oryzae
Rhizopus oligosporus
Rhizopus stolonifer
Scopulariopsis brevicaulis
Scopulariopsis candida
Scytalidium lignicola
Trichoderma viride
Trichothecium roseum
Ulocladium consortiale
Cows
n = 20
Pigs
n = 20
Rabbits
n = 15
1
—
1
—
—
2
1
1
—
1
—
—
—
1
—
3
—
—
—
—
—
—
—
1
2
—
—
2
—
1
Chickens
n = 15
Statistically significant differences (% test) p < 0.05.
** Statistically significant différentes (%^ test) p < 0.01
companying fungal flora, strains of the genera Alternaría, Aspergillus, Acremonium, Cladosporium,
Geotrichum, Pénicillium and Scopulariopsis. Hubalek et al. (6), in a survey of the fungi isolated on
the hair of small wild mammals reported a high incidence of different species belonging to the genera Pénicillium, Aspergillus and Alternarla. Aho (1) examined hair samples of dogs, cats, horses,
cows, rats and other animals for the presence of saprophytic fungi. The commonest in order of frequency were members of the genera Pénicillium, Cladosporium, Aspergillus, Mucor, Aureobasidium. Alternaría, Scopulariopsis, Trichoderma and Trichothecium. Statistically significant differences in contamination of animal skin were noted in some cases.
In our study, the genus Pénicillium was isolated significantly more often (x^ test p < 0.01) from
chicken samples than those of rabbits, pigs or cows. P. chrysogenum and P. viridicatum were the
most frequent species of Pénicillium isolated.
The commonest species of Aspergillus isolated were A. fumigatus, A. candidus, and A. tubingensis. Statistically significant differences {y^ test p < 0.05) were noted in A. tubingensis, more frequent in cow's hair.
Alternarla alternata, one of the most frequent species in the study was isolated significantly more often (%^ test p < 0.01) from rabbit samples than those of cows, pigs or chickens.
We have to point out the isolation of Arthroderma sp., perfect state of Trichophyton mentagrophytes in two rabbit's hair samples without apparent injury. The dermatophyte
T. mentagrophytes is one of the most common causes of tinea in rabbits, and it has been isolated
from healthy animals as wild rodents (10, 12, 13), laboratory animals (3, 4, 5, 9), bird feathers (7)
and dogs (2), suggesting that in some cases, animals can act as healthy carriers. They do represent a
potential risk of infection both for other animals living in the same animal house and for the people
working with them.
The hair of animals serves as a collector of environmental fungi and thus it acquires a transient
fungal flora. In this study the fungal flora isolated from the skin of the animals reflected in general
the abundance of fungi in their environments because the samples were taken inside the farmhouses.
It is unknown how long the fungi can survive on the skin of animals and how some fungi can
multiply on the coats of animals contaminated by soil and foodstuffs. The presence of opportunitic
fungi on the coats of animals creates an opportunity for them under special circumstances to become invasive and thus to cause primary or secondary infections. Because of that, further studies are
obviously required.
M." L. LAB ARCA, M.' R. BRAGULAT, M.' T. BRUGUERA AND F. J. CABANES
125
References
1. Aho, R. (1983). Saprophytic fungi isolated from the hair of domestic and laboratory animals with suscepted dermatophytoses. Mycopathologia 83,65-73.
2. Cutsem, J. van, de Keyser, H., Rochette, F. and vand der Flaes, M. (1985). Survey of fungal isolates from alopécie and
asymptomatic dogs. Vet. Rec. 116,568-569.
3. Feuerman, E., Alteras, I., Honig, E. and Lehrer, N. (1975). Saprophytic occurrence of Trichophyton mentagrophytes and
Microsporum gypseum in the coats of healthy laboratory animals. Mycopathologia 55,13-15.
4. Fischman, O., de Camargo, Z. P. and Grinblat, M. (1976). Trichophyton mentagrophytes infection in laboratory white mice. Mycopathologia 59,113-115.
5. Hironaga, M., Fujigati, T. and Watanabe, S. (1981). Trichophyton mentagrophytes skin infections in laboratory animals as
a cause of zoonosis. Mycopathologia 73,101-104.
6. Huabalek, Z., Rosicky, B. and Otcenasek, M. (1979). Fungi on the hair of small wild mammals in Czechoslovakia and Yugoslavia. Ces. Mykol. 33,81-93.
7. Humpolickova, V. and Otcenasek, M. (1981). Keratinophilic fungi from the feathers of free-living birds. Folia Parasitol.
28,179-186.
8. Kushida, T., Imamura, K., Kuwahara, S., Kuwahara, H., Shimada, Y., Takahashi, T., Tahara, S., Nishimura, K. and Yamada, T. (1972). Keratinophylic fungi on hair of dogs and cats wihtout visible skin lesions in Kyoto. J. Jap. Vet. Med, Ass.
32,552-554.
9. López Martínez, R., Mier, T. and Quirarte, M. (1984). Dermatophytes isolated from laboratory animals. Mycopathologia
88,111-113.
10. Mariât, E, Châtelain, J. and Rouffaud, M. A. (1976). Etude sur la contamination par les champignons dermatophytes
d'une population de petits mammifères sauvages en Alsace. Mycopathologia 58,71-78.
11. Marsella, R., Mercantini, R., SpineUi, R and Volterra, L. (1985). Occurrence of keratinophihc fungi in animais of the zoological park of Rome. Mykosen 28,507-512.
12. Otcenasek, M., Hubalek, Z. and Sixl, W. (1980). Survey of dermatophytes in the hair of small mammals in AustraUa. Folia Parsitol. 27,83-87.
13. Ozegovic, L. (1980). Wild animals as reservoirs of human pathogenic dermatophytes. Med. Mycol. Zbl. Bakt. Suppl. 8,
369-380.
14. Philpot, C. M. and Berry, A. P. (1984). The normal fungal flora of dogs. A preliminary repport. Mycopathologia 87,155157.
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M I C R O B I O L O G Í A SEM 5<1989),
127-128
NOTES
A rational proposal for plasmid nomenclature
p. Campos* and F. Martín Luengo
Departamento de Genética y Microbiología. Facultad de Medicina. Universidad de Murcia. Campus Universitario de Espinardo.
Murcia, Spain.
(Received March 9/June 6,1989)
Summary
We propose a more rational system for nomenclature of wild plasmids of bacteria.With this
proposal for nomenclature of bacterial plasmids, it is estabhshed in an unambiguous way: 1) if a
plasmid is wild or derivative, and 2) in which species and bacterial strain it was found (in the case of
wild plasmids).
Key words: Plasmid nomenclature.
Resumen
Nosotros proponemos un sistema más racional para la nomenclatura de los plásmidos salvajes
de las bacterias. Con esta proposición para la nomenclatura de los plásmidos de las bacterias se establece sin ambigüedades: 1) si un plásmido es salvaje o derivado, y 2) en qué especie y cepa bacteriana fue encontrado (en el caso de los plásmidos salvajes).
Biological nomenclature has as a principal objective to name organisms/molecules in a very
specific and unambiguous way, following internationally recognized rules. In bacterial nomenclature, following the binomial system, the names of bacterial species are necessary and must be stable
and unambiguous (6). With this system, new bacterial species can be introduced in bacterial taxonomy in an unambiguous way. In molecular nomenclature, unambiguous names have been proposed for the several molecular species in a logic way. In the case of restriction endonucleases (5), these enzymes have the general name of restriction endonucleases, followed by a code of three letters
(formed by the first letter, in capital letter, of the genus name, and the two first letters, in small letters, of the specific epithet of the bacterial species that presents a particular modification-restriction
system), and the name of the bacterial strain and/or a serial number that identifies a particular bacterial strain. For example, the restriction endonuclease R. Eco RI means: «R» of restriction endonuclease, «E» oí Escherichia, «co» of coliy and «RI» of first endonuclease codified by a resistance
plasmid (plasmid RY13) in E. coll With this system of restriction endonuclease nomenclature, it is
not ambiguous to name the new endonucleases that are continually being isolated (1).
Bacterial plasmids, in their first period, were named: «F» (fertility factor), «Col» followed by
(*) Corresponding author.
128
A RATIONAL PROPOSAL FOR...
an identification (for bacteriocinogenic plasmids of E. coli) and «R» followed by an identification
number (for resistance plasmids). Due to the isolation of new plasmids, this system was unacceptable. In 1976, a system of nomenclature for bacterial plasmids was proposed (3). In this system it was
recommended to use the letter «p» to designate a plasmid, followed by a prefix (two/three/four letters) to identify a particular investigator/laboratory and a serial number of that investigator/laboratory. With this system, plasmid pXY1234 means: plasmid number 1234 from the collection of the investigator/laboratory using the prefix/initials of identification XY. A great number of prefixes have
been registered for plasmid designations to avoid undesirable dupUcations (2). This system of nomenclature for bacterial plasmids is completely unacceptable, due to: 1) it does not distinguish between wild plasmids and derivative plasmids (mutants, recombinants, etc.); 2) it does not offer information about the primary natural host of plasmids, and 3) the prefix offers neither information nor
reability due to plasmids are being isolated from many bacterial species (throughout the world) and
continually new derivative plasmids are being made [for example, pHH is used to name plasmids of
Hammersmith Hospital (London) (3) and plasmids of Halabacterium halobium (4)]. Therefore, this
system of nomenclature for bacterial plasmids tends towards a future collapse, because it does not
permit the introduction of new plasmid names in an indefinite and unambiguous way.
Until very specific and unambiguous rules are developed, we propose here a more rational system for plasmid nomenclature. In this proposal, we put forward that the nomenclature for wild plasmids must be different from the nomenclature for derivative plasmids (mutants, recombinants,
etc.). In the case of derivative plasmids, we propose the nomenclature already established in 1976
(3), until a better solution be found. In the case of wild plasmids of bacteria, we propose that they
be'named: «p» to designate a plasmid, followed by a code of three letters (formed by the first letter,
in capital letter, of the genus name, and the two first letters, in small letters, of the specific epithet of
the bacterial species where the plasmid was found) as in the system of restriction endonucleases nomenclature, followed by a hyphen and the name of bacterial strain (not a prefix and a serial number
of the plasmid). The bacterial strain could be designated by a prefix of the investigator/laboratory
followed by a serial number or a prefix of a reference collection of bacteria followed by a serial
number. If the bacterial strain has more than one plasmid, the designation of the strain could be followed by «A», «B», «C», etc., in order to designate plasmids from larger to smaller molecular weight.
With this new system, plasmid pEco-XY1234A means: first wild plasmid (in molecular weight) of
the bacterial strain XY1234 (whose prefix/initials of identification is XY and whose serial number is
1234) of Escherichia coli. Principally, this proposal for nomenclature of bacterial plasmids establishes in an unambiguous way: 1) if a plasmid is wild or derivative (for example, pEco-XY1234A is a
wild plasmid and pXY1234 is a derivative plasmid), and 2) in the case of wild plasmids, in which
species and bacterial strain it was found (for example, pEco-XY1234A is the first wild plasmid of
the strain XY1234 of Escherichia coli).
References
1. Kessler, C. and Holtke, H. J. (1986). Specificity of restriction endonucleases and methylases. A review. (Edition 2.) Gene
47,1-153.
2. Lederberg, E. M. (1986). Plasmid prefix designations registered by the Plasmid Reference Center 1977-1985. Plasmid 15,
57-92.
3. Novick, R. R, Clowes, R. C , Cohen, S. N., Curtis III, R., Datta, N. and Falkow, S. (1976).Uniform nomenclature for bacte• rial plasmids: a proposal. Bacteriol. Rev. 40,168-189.
4. Pfeifer, R, Weidinger, G. and Goebel, W. (1981). Characterization of plasmids in halobacteria. J. Bacteriol. 145 369-374.
5. Smith, H. O. and Nathans, D. (1973). A suggested nomenclature for bacterial host modification and restriction systems
and their enzymes. J. Mol. Biol. 81,419-423.
6. Sneath, P. H. A. (1984). Bacterial nomenclature. In: N. R. Krieg et al. (eds,). Bergey's Manual of Systematic Bacteriology,
vol. 1, pp. 19-23. Williams and Wilkins, Baltimore.
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